The production of artificial antigens with good immunogenicity is the most critical step in establishing immunoassay methods for determining small molecule compounds. The design and modification of hapten (selection of modification site, selection of modification method, selection of linker), selection of carrier, coupling of hapten and carrier (coupling mode, coupling condition), purification and identification of artificial antigen are introduced as follows.

Immunoassay is a trace analysis method based on the specific recognition and reversible binding reaction between antigen and antibody, which has been widely used in clinical analysis and biological analysis. Immunoassay is not only suitable for the detection of large molecular compounds (such as proteins, nucleic acids, and bacteria), but also for the determination of small molecular compounds (such as hormones, drugs). Because immunoassay has the advantages of strong specificity, high sensitivity, simplicity, rapidness, low cost, and suitability for screening large quantities of samples in the field, its application in environmental and food safety analysis has attracted much attention. In recent years, immunoassay methods have been used for pesticides, veterinary drugs, environmental hormones, toxins, and banned food additives in environmental and food samples. The key to the establishment of immunoassay methods for small molecule compounds is to prepare antibodies with high affinity and selectivity for small molecule compounds. Since most pesticides and veterinary drugs are small molecule compounds (molecular weight less than 1000), they do not have immunogenicity. That is, they lack T cell epitopes and cannot directly induce specific antibodies in the animal body, so small molecule compounds are called haptens. However, the haptens-carrier complex can be formed by means of appropriate chemical modification at a certain position of its divided structure, the linker with the active group as the upper group, and then combined with macromolecular carriers. The conjugate of the carrier, that is, the artificial antigen, can indirectly induce the proliferation and differentiation of B cells by means of T cell epitopes and produce specific antibodies. Therefore, hapten design (that is, how to select the modification site of hapten), hapten modification (that is, how to synthesize a functional linker with a certain length), and artificial antigen production (that is, hapten-linker and carrier coupling) are important processes for the production of anti-small molecule compound antibodies and the establishment of corresponding immunoassay methods.

Design of Haptens

In order to prepare antibodies against small molecule compounds, there are certain requirements for the molecular structure of the hapten itself. There should be a certain complexity or rigidity, such as containing benzene rings, heterocyclic groups, or containing branch structures, otherwise, it is difficult to produce antibodies, or the resulting antibodies' titer is low. Studies have shown that if the hapten molecule has a benzene ring, the success rate of antibody production is 1/3, and the success rate without a benzene ring is 1/11.

If the hapten molecule has active groups, such as -COOH, -NH, -OH, the hapten can be directly conjugated with the carrier to produce artificial antigen under the premise that the structure of hapten itself can basically meet the requirements for antibody production. If the hapten has no groups that can be directly covalently conjugated to the carrier or has active groups but these groups are very important for maintaining the immune properties and characteristic structure of the hapten, or the characteristic structure of the hapten after being directly connected to the carrier is susceptible to the interference of the local microchemical environment or steric hindrance of the carrier, affecting the recognition of the body's immune system, then the hapten must be redesigned.

The basic requirement of hapten design is to keep the original molecular characteristic structure of hapten as far as possible so that it can be exposed to the surface of the artificial antigen so that it can be recognized by the animal immune active cells to the maximum extent, to stimulate the body to produce a specific immune response and produce antibodies with high affinity and high specificity for the test object. Therefore, the key to hapten design is to determine the appropriate modification site in the hapten structure and the connection with a certain carbon chain length and the active end group at the modification site by chemical method.

Choice of Linker

The connecting part between the hapten characteristic molecular structure and the carrier is the linker. The main purpose of introducing the linker is to highlight the characteristic structure of haptens (generally important antigenic determinant) on the surface of artificial antigens to facilitate the production of antibodies with high specificity.

The selection of the linker should follow the following basic principles: (1) The connection of the linker should be avoided at or near the functional group of the target hapten, and it is best to be located at the far end of the important characteristic functional group to avoid reducing the recognition of the antibody and the target hapten. For a class of compounds with similar structures, the linker should be connected at the position where they have the same structure (or similar structure) to expose the characteristic parts of the molecule to the maximum extent; (2) The length of the linker should be appropriate. If the linker is too short, the steric hindrance of the carrier affects the recognition of hapten characteristic structure by the animal immune system, and the stereostructure of hapten is easy to change under the influence of the local chemical environment of the carrier. If the linker is too long, the hapten may be "folded" by hydrogen bonding (some polar linkers), hydrophobic action (non-polar linkers), or other forces. The optimum length of the linker may be different for different haptens. (3) Avoid linkers containing strong determiners (such as aromatic rings, co-giant double bonds, or heterocyclic rings), and usually use chain hydrocarbons containing terminal active groups as appropriate to reduce the generated antibodies to over-recognition of linkers and reduce the recognition ability of target molecules.

Carrier Selection

In artificial antigen, the carrier can not only increase the relative molecular mass of hapten or only play the role of transport but also rely on its own structural specificity and immunogenicity to induce the body to produce an immune response and then induce the recognition of hapten molecules, this phenomenon is called carrier effect. In the immune response, B cells recognize the hapten determinant family and T cells recognize the carrier determinant family.

Fig 1 T-B collaboration & the hapten-carrier effect

Common carriers are proteins such as Globulin fractions, Bovine serum albumin (BSA), Ovalburmn (OVA), Keyhole limpet hemo-cyanin (KLH), Rabbit serum albumin (RSA), Human serum albumin (HSA), Thyroglobulin, Fibrinogen, or rabbit and chicken gamma globulin. Among them, the most common carrier is BSA. KLH is considered the preferred carrier because of its good heterogenicity with the immune system of holospondylus, but KLH is expensive. In recent years, it has also been reported that synthetic skin (commonly used polylysine PLL) is used as a carrier, which has the advantage of increasing the immunogenicity of haptens.

Coupling of Hapten and Carrier

The commonly used methods for coupling hapten with a carrier are chemical coupling methods, chemical-biological methods, and mmunological labeling methods. Among them, the chemical coupling method is the most commonly used, which uses chemical reagents (coupling agents) to connect two substances under certain conditions (one-step method or direct method).

Conjugated Hapten-linker and Protein

Generally, according to the different active groups contained in the hapten, different ways can be selected to couple with the carrier protein. If hapten contains antelope group, it can be coupled with carrier protein by carbodiimide method, mixed anhydride method, active ester method; if hapten contains amino group, it can be coupled with carrier protein by glutaraldehyde method, halogenated nitrobenzene method, diazotization method. If hapten contains hydroxyl group, it can be coupled with carrier protein by azobenzoic acid method, sodium monochloroacetate method. If hapten contains sulfhydryl group, it can be coupled with carrier protein by disulfide bond through SAMSA(S-acid group succinic acid) reaction. If the hapten contains an aldehyde group or a ketone group, an intermediate with a carboxyl group can be synthesized by the O-(antelope methyl) hydroxylamine method and p-hydrazine benzoic acid method, and then the carboxyl group is bound to the amino group of the protein.

Fig 2 Coupling methods

Coupling Conditions of Hapten-linker and Carrier Protein

The coupling conditions of hapten and carrier protein mainly include: (1) the relative concentration and initial molar ratio of hapten, carrier protein and coupling agent in the coupling reaction; (2) Buffer composition, pH(general pH=6.0-8.0) and ionic composition for dissolving hapten, carrier protein and coupling agent; (3) If hapten and coupling agent can be dissolved in water, the coupling reaction can be carried out in the water phase; if hapten in water solubility is not large or insoluble (such as steroid hormones and other fat soluble substances), then the coupling reaction should be carried out in the organic phase, at this time, organic solvents that have no effect on the biological activity of carrier proteins (such as pyridine, dioxane, acetone, dimethylformamide) should be selected to dissolve the hapten and keep the carrier protein at soluble state; (4) The temperature and time of the coupling reaction should be properly controlled, and finally, special protective measures should be taken for hapten or carrier proteins in some special coupling reactions.

Purification and Identification of Artificial Antigens

The artificial antigens obtained after the hapten is coupled to the carrier must be purified to remove unreacted hapten small molecules, salts, and other small molecular impurities. The most common methods are dialysis and gel chromatography. Dialysis generally takes a long time (usually more than 2d), the purification is more thorough, and the operation is relatively simple. Gel chromatography requires a short time, but the operation is relatively complex, and the efflux component needs to be tracked and analyzed to determine the target component.

On the one hand, the identification of purified artificial antigens is to judge whether hapten and carrier coupling is qualitatively successful. On the other hand, the coupling rate and protein content were quantitatively determined. The most commonly used method to determine whether the coupling is successful is ultraviolet scanning. If the ultraviolet absorption characteristics of the artificial antigen have the ultraviolet absorption characteristics of both the hapten and the carrier protein, the initial judgment of the coupling success can be made. The coupling rate was determined by ultraviolet spectrophotometry, labeled antigen tracer, SDS-PAGE, ESI-MS, MALDI-TOF-MS, and so on. For organophosphorus pesticides, it can be determined by the method of determining the ratio of phosphorus concentration and protein concentration, such as the determination of methyl-p-oxy-phosphorus binding ratio or the calculation of the difference in the number of free -NH2 in the protein before and after the coupling reaction, such as dinitrophenol method; Or according to the change of amino acid content before and after the reaction; There are also elemental analysis and infrared spectroscopy methods.