In vitro diagnosis is an important source of clinical diagnostic information and an indispensable part of the medical system to ensure human health. Antigens, antibodies, and diagnostic enzymes are the core raw materials in the upstream of the in vitro diagnostic reagent industry. The quality of small molecule antigen raw materials is also an important factor in determining the quality of in vitro diagnostic reagents.

New Core Raw Materials—Small Molecule Detection Raw Materials

Diagreat Biotechnologies Co., Ltd has built a mature R&D and production platform for core raw materials of in vitro diagnostic reagents through independent innovation. The artificial modification of the small molecule hapten greatly improves its immunogenicity while retaining the characteristic epitope of the hapten, which improves the preparation process of the hapten antibody. Antigen products are one of Diagreat’s best- selling high-quality products and have a good reputation. The products are mainly used in reference standards, calibrators, quality controls, and immunogens. Diagreat takes the core raw material supplier of professional in vitro diagnostic reagents as its own responsibility, continuously improves the product system, deeply expands the research and development of raw materials, actively embraces the global market, and promotes the wide application of high-quality poducts at home and abroad.

Raw materials for small molecule detection

Other detection raw materials

Small molecule antigens refer to small molecule compounds with generally small molecular weights. Generally speaking, the larger the molecular weight and the more complex the structure, the easier it is to prepare high-affinity antibodies, and small molecules containing aromatic rings are more likely to cause immune reactions. Similarly, small molecule compounds must be coupled with carrier proteins to form complete antigens as immunogens. Coupling of small-molecule compounds with carriers generally requires the introduction of some linking arms or linkers through intermediate pathways and then connected to protein carriers, which generally cannot be directly coupled to a certain group on its structure. If the coupling is forced, the structure of the antigen will be changed. Therefore, the complete antigen is usually prepared by synthesizing intermediates first.

Small molecule hapten activity is affected by multiple factors

Including the influence of the original molecular structure, linking arm, active group, and spatial conformation, many technical difficulties will be encountered during the preparation process. The common technical difficulties include the following aspects:

1) The selection of the antigen linking site, the antigen immune activity obtained by modifying different sites may vary greatly.

2) The selection of the antigen linking arm and its length, the linking arm and the additional active group should not easily induce the body to produce “arm antibody”.

3) Judgment of the connection effect between the small molecule hapten and the carrier protein, such as the judgment of the binding ratio and coupling rate.

4) Judgment of successfully prepared artificial antigen concentration and the strength of immunogenicity.

5) The specificity and affinity of the prepared antibody.

Hapten Design and Synthesis

The purpose of hapten design is to make the hapten stimulate the body to produce a specific immune response, and to obtain antibodies with high affinity to the analyte molecule. 1) The hapten in the immunogen should be as similar as possible to the analyte molecule in terms of molecular structure, stereochemistry and electronic distribution. 2) The linking arm in the hapten structure should not be easy to induce “arm antibody”, it is best to use A certain length of carbon chain. 3) The hapten molecule should have active groups (such as -NH2, -COOH, -OH, -SH, etc.) that are convenient for coupling with protein carriers, and the presence of active groups should have no effect on the electron distribution of the analyte molecule. 4) The basic structure of the analyte molecule should still be retained after the hapten is coupled to the protein.

1. Introduction of linking arm

In order to highlight the characteristic structure of the analyte molecule, the hapten design often introduces a certain length of linking molecule between the characteristic structure and the carrier protein, that is, the linking arm. Introducing a linking arm is a more commonly used solution for the preparation of hapten antibodies. It has been reported that when a small molecule hapten antibody is prepared, it is found that when a shorter linking arm is used or no linking arm is used, the antibody against the hapten cannot be induced, while the hapten with a longer linking arm can induce the production of antibodies against the hapten. The explanation for this phenomenon is that when the linking arm is short or there is no linking arm, the hapten may be covered by the three-dimensional structure of the carrier protein, and when the linking arm is long enough, it is beneficial for the small molecule hapten to be fully exposed to the surface of the carrier protein, which facilitates its recognition by antigen-presenting cells. It is generally believed that the optimal length of the linking arm is between 3-6 straight-chain carbon atoms. Too short linking arm is not conducive to the full exposure of the hapten, while too long linking arm will cause the folding of the alkyl chain due to hydrophobic interaction. As a result, the hapten molecule is still covered by the carrier protein, which is not conducive to the recognition of antigen-presenting cells. In addition, studies have suggested that the use of linking arms with different structures, different coupling methods or changing the introduction position of the linking arms are also beneficial to the production of antibodies and improve the affinity and binding force of antibodies.

2.Introduction of active groups

There are two ways to introduce active groups: one is to use the existing active groups (-NH2, -COOH, -OH, -SH, etc.) on the analyte molecule, with the help of bi-functional reagents (such as NH2(CH2)nCOOH, SH(CH2)nCOOH, succinic anhydride, glutaraldehyde, etc.), to introduce linking arms and active groups to couple with carrier proteins. The advantage of this method is that it is relatively easy to implement, but for some analytes, if these active groups are their characteristic structures, or the electron distribution of the molecule is changed after coupling, it will affect the recognition of the analyte by the antibody. Another method is to directly synthesize derivatives with structures such as -(CH2)nCOOH, -(CH2)nNH2 of the analyte, which is beneficial to protect the characteristic structure of the analyte and the electron distribution of the molecule from being affected , but the synthesis is sometimes difficult and often requires multi-step reactions to achieve.

3.Use of heterogeneous haptens

The use of heterogeneous haptens is an indirect method to increase the affinity of antibodies to the analyte. In recent years, some researchers have proposed the concept of hapten design using the “steric hindrance effect”. It was found that by introducing different degrees of steric hindrance into the haptens of artificial immunogens, the induced polyclonal antibodies can selectively recognize different sizes of molecules in a series of homologues. In addition, studies have shown that in competitive immunological detection methods, the hapten structure with large steric hindrance can reduce the binding ability of the coating source or enzyme label to the antibody, so as to improve the affinity of the free analyte to the antibody, and the sensitivity and specificity can be greatly improved.

4. Selection of hapten carrier protein

Since most drugs, toxins, environmental pollutants and other hapten substances have no immunogenicity, they usually need to be coupled with a large molecular weight carrier protein to prepare a complete antigen (immunogen) and obtain immunogenicity with the help of T cell epitope of the carrier protein, so as to stimulate the body to produce antibodies. Carriers commonly used in the preparation of complete antigens include bovine serum albumin (BSA), chicken ovalbumin (OVA), keyhole limpet hemocyanin (KLH), rabbit serum albumin (RSA), human serum albumin (HSA), Polylysine (PLL), etc. In addition, to obtain a better immune effect, immune adjuvants are often used.