This work was supported by the National Natural Science Foundation of China (grant numbers 81573573, 81473338, and 81503344) and the Classical Prescription Basic Research Team at the Beijing University of Chinese Medicine.

All of the animal procedures performed in this study have been approved by the Ethical Review Committee at the Beijing University of Chinese Medicine (approval number 2016BZYYL00109).
NOTE: Female BALB/c mice (8 weeks old) were immunized with hapten-carrier protein conjugates. When used alone, a small molecule (&#60;1,000 Da) cannot elicit an immune response. However, conjugating the small molecule to a carrier macromolecule results in antigen synthesis. In this context, the small molecule is ...

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The authors have nothing to disclose.

Here, we present a protocol for the successful production of mAbs against natural product-derived small molecules. The essential steps in the procedure have been outlined, and we have demonstrated the utility of this protocol using NAR as an example small molecule. The example spectra, reactivity analyses, and icELISA results all show representative experimental and control data that is obtained using this protocol. Example images of the hybridomas provide a visual representation of what the researcher should be looking ...

Monoclonal antibodies (mAbs), also known as mono-specific antibodies, are produced from a single B-lymphocyte clone and are composed of monovalent antibodies that all bind to the same epitope1. In recent years, many medicinal plant-derived natural products have been used in the treatment of various diseases2. Indeed, many small molecular compounds originally derived from natural products are now applied as first-line drugs, such as artemisinin for malaria and paciltaxel (taxol) for cancer2,3. The study of natural products has made rapid progress, largely due to the tremendous development and optimization of conventional analysis techniques, including high performance liquid chromatography (HPLC) and mass spectrometry (MS). However, there are still some limitations associated with these methods, such as their complex pretreatment protocols and associated costs with regards to time, labor/expertise, and required instruments4.
Recently, mAb-based enzyme-linked immunosorbent assays (ELISAs) have been applied to qualitatively and quantitatively analyze food and natural products. In fact, this method has been applied for both biological samples analysis and clinical testing and has been shown to be accurate, sensitive, and highly efficient while also avoiding the tedious pretreatment steps associated with other analyses5,6.
When using mAb-based ELISAs to study complex natural products, preparation of the monoclonal antibodies is one of the core steps. Unfortunately, the mAbs specific to the small bioactive components present in these types of substances6,7,8,9,10,11,12,13,14,15 are often limited compared to the protein antigens. To circumvent this issue, we have developed a protocol to specifically generate mAbs against small compounds. The protocol presented here includes artificial antigen synthesis, mouse immunization, cell fusion, indirect competitive ELISA, and monoclonal hybridoma preparation.
Notably, our research group has been studying the formation of mAbs against small bioactive compounds from traditional Chinese medicines and developing their applications for years. In our on-going studies, we have developed mAbs against baicalin16, puerarin17, glycyrrhizic acid18, paeoniflorin19, ginsenoside Re20, ginsenoside Rh121, and many other small molecules. Our ELISA protocols based on these mAbs have been used in a number of studies to evaluate the pharmacokinetics of these small molecules as well as their interactions with other bioactive compounds. Moreover, using these mAbs, we have also developed immunoaffinity chromatography methods for the separation of structural analogues, including epimers. Recently, we prepared a lateral flow immunoassay using our anti-puerarin mAb that was subsequently used for rapid, on-site detection of this compound. Our results indicate that our mAb-based assays are indispensable and convenient tools for studying the biology and quality of natural-product-derived compounds, particularly those used in traditional Chinese medicines.

<table border="0" cellpadding="0" cellspacing="0" style="width:619px;" width="617">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">800 mesh (40 &#956;m nylon) filter&#160;</td>
			<td style="width:101px;">FALCON</td>
			<td style="width:123px;">352340</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">24 well culture plate</td>
			<td style="width:101px;">NUNC</td>
			<td style="width:123px;">119567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">25 cm2 Flask</td>
			<td style="width:101px;">Labserv</td>
			<td style="width:123px;">310109016</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">3,3&#8217;,5,5&#8217;-Tetramethylbenzidine(TMB)</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">860336 1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">75 cm2 Flask</td>
			<td style="width:101px;">Corning</td>
			<td style="width:123px;">430720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">96 well culture plate</td>
			<td style="width:101px;">NUNC</td>
			<td style="width:123px;">117246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">bovine serum albumin</td>
			<td style="width:101px;">AMRESCO</td>
			<td style="width:123px;">332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">cell strainer</td>
			<td style="width:101px;">FALCON</td>
			<td style="width:123px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">centrifuge tube 15 mL</td>
			<td style="width:101px;">Corning</td>
			<td style="width:123px;">430645</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">centrifuge tube 50 mL</td>
			<td style="width:101px;">Corning</td>
			<td style="width:123px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">cryotubes, 1 mL&#160;</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">V7384-1CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">cultivator</td>
			<td style="width:101px;">DRP-9082&#160;</td>
			<td style="width:123px;">Samsung</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">dialysis membrane (10kDa)</td>
			<td style="width:101px;">Heng Hui</td>
			<td style="width:123px;">45-10000D</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">dimethylsulfoxide</td>
			<td style="width:101px;">Sinopharm Chemical</td>
			<td style="width:123px;">DH105-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">electronic balance&#160;</td>
			<td style="width:101px;">BS124-S&#160;</td>
			<td style="width:123px;">Sartorius</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">ELISA plates, 96 well</td>
			<td style="width:101px;">NUNC</td>
			<td style="width:123px;">655101</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">ethanol, 96%</td>
			<td style="width:101px;">Sinopharm Chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal bovine serum</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">fetal calf serum</td>
			<td style="width:101px;">Invitrogen</td>
			<td style="width:123px;">10270106</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Freund&#180;s adjuvant, complete</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">SLBM2183V</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Freund&#180;s adjuvant, incomplete</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">SLBL0210V</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Gelatin</td>
			<td style="width:101px;">AMRESCO</td>
			<td style="width:123px;">9764-500g</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Gradient cooler container</td>
			<td style="width:101px;">Nalgene</td>
			<td style="width:123px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">HAT media supplement</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">H0262-10VL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">HRP-conjugated goat-anti-mouse IgG antibody</td>
			<td style="width:101px;">applygen</td>
			<td style="width:123px;">C1308</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">HT media supplement</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">H0137-10VL</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Inverted Microscope</td>
			<td style="width:101px;">IX73</td>
			<td style="width:123px;">Olympus&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">keyhole limpet hemocyanin</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">H8283</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">MALDI-TOF-MS&#160;</td>
			<td style="width:101px;">Axima-CFR&#160; plus&#160;&#160;</td>
			<td style="width:123px;">Axima&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Microplate Reader</td>
			<td style="width:101px;">BioTex</td>
			<td style="width:123px;">ELX-800&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">mouse</td>
			<td style="width:101px;">Vital River&#160;</td>
			<td style="width:123px;">BALB/c</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">ovalbumin</td>
			<td style="width:101px;">Beijing BIODEE</td>
			<td style="width:123px;">5008-25g</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">PEG</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:123px;">RNBC6325</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Penicillin&#38;Streptomycin solution</td>
			<td style="width:101px;">Hyclone</td>
			<td style="width:123px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Pipette 10 mL</td>
			<td style="width:101px;">COSTAR</td>
			<td style="width:123px;">4488</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">Pipette 25 mL</td>
			<td style="width:101px;">FALCON</td>
			<td style="width:123px;">357525</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:223px;">RPMI 1640</td>
			<td style="width:101px;">Corning</td>
			<td style="width:123px;">10-040-CVR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">skim milk</td>
			<td style="width:101px;">applygen</td>
			<td style="width:123px;">P1622</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:223px;">sodium periodate</td>
			<td style="width:101px;">Sinopharm Chemical</td>
			<td style="width:123px;">BW-G0008</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:223px;">Sulfo-GMBS</td>
			<td style="width:101px;">Perbio Science Germany</td>
			<td style="width:123px;">22324</td>
			<td></td>
		</tr>
		<tr height="19">
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generation of monoclonal antibodies against natural products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

Generation of monoclonal hybridomas
The molecular weight of the hapten-carrier conjugate was confirmed by MALDI-TOF-MS analysis. As the molecular weight of both BSA and the NAR are known, the number of small molecules conjugated with BSA could be calculated. Figure 1 shows representative spectral results for NAR-BSA22, which displays a broad ...

Watch this Scientific Journal Video about Generation of Monoclonal Antibodies Against Natural Products at JoVE.com

Generation of Monoclonal Antibodies Against Natural Products

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Monoclonal antibodies are known as mono-specific antibodies which are produced from a single B-lymphocyte clone and the monovalent antibodies which bind to the same epitope. Recently, the monoclonal antibody-based ELISA has become our emerging platform for the qualitative or quantitative analysis of foods or natural products. This method is a accurate, sensitive, and effective method without tedious pretreatment steps.So that is the essential for biological samples and the future clinical applications. Preparation of the mABs of TCM is a vital step in studies on complex system characteristics of TCM formula. We have developed a protocol to generate mABs against the small molecular compounds, including artificial antigen synthesis, mouse immunization, and cell fusion.The hapten is connected with the carrier protein to synthesize the artificial antigen. Artificial antigen and complete adjuvants are mixed and emulsified. The mouse was immunized by subcutaneous injection.Take out the spleen of immunized mouse and make single-cell suspension. Mix the myeloma cells. Splenocytes are isolated and fused with HAT-sensitive mouse myeloma cell line by PEG method.Select a cell line able to prepare antibodies by ELISA. Hybridomas secreting monoclonal antibodies that are reactive to antigen are cloned. Establish the hybridoma cell line is cryopreserved.Periodate oxidation procedure was used for the synthesis of a narigin BSA conjugate. Firstly, narigin was dissolved at a final concentration of one milligram per milliliter at 100 microliters of freshly prepared sodium pariodate solution to five milliliters of the narigin solution. Stir the mixture at room temperature for one hour.Secondly, add two milliliters of carrier protein to the narigin sodium periodate reaction mixture. Adjust the mixture to pH nine solution and continue to react for six to eight hours. Thirdly, the artificial antigen was purified with dialysis method in PDS for three days to estrange the CBS.Freund's complete adjuvant and incomplete adjuvant were used for mouse immunization. For the vaccination, mix 50 micrograms of conjugate with an equal volume of Freund's complete adjuvant and emulsify completely. Administrate 100 micrograms of this mixture to each BALB/c mouse via dorsal subcutaneous injection.Deliver a booster vaccination via subcutaneous injection two weeks later with the same amount of conjugate mixed with incomplete adjuvant. For primary immunization, Freund's complete adjuvant and immunogen were used via subdermal injection. For booster immunization, Freund's incomplete adjuvant and the immunogen were used via subdermal injection.For impact immunization without adjuvant, only immunogen were used via subdermal injection or intraperineal injection. Feed layer cells mainly originated from abdominal epithelial cells or macrophage. RPMI 1640 FBS and HAT were used for preparing HAT screen medium.HAT supplement was dissolved in 10 milliliters RPMI medium, then added into 500 milliliters RPMI 1640 containing 20%FBS. And ICR mouse was sterilized by 75%ethyl alcohol for five minutes. After removing the fur from the abdomen with hemostatic forceps, disinfect area with 75%ethanol.Cut the outer skin to expose the abdominal cavity. Inject three milliliters of sterile RPMI 1640 medium into the abdomen. Then massage the abdomen to detach additional cells into the solution.Aspirate the fetal cell suspension into a 15 milliliter centrifuge tube. After centrifuging the cell suspension for 10 minutes at 1, 000g, discard the supernatant and re-suspend the fetal cells to get single cell suspension. After this procedure, dissolve the cells by using HAT medium.Count the cells to make it 100, 000 per milliliter from cell members. Transfer the cells into 96 well cell culture plates. Incubate the plates overnight at 37 degree, 5%carbon dioxide.The chosen immunized mouse was sterilized by 75%ethyl alcohol for five minutes. Aspirate RPMI 1640 medium as the washing buffer. Remove the skin and muscle tissue using scissors to expose the spleen.Isolate the spleen by the tweezers carefully. Then wash the spleen in the RPMI 1640. Isolate the spleen and cut into pieces.Slowly pound the spleen. Then it was treated with an RMPI 1640 medium to dissolve the cells to prepare a spleen cell suspension. After that, the cells were filtered by 800 mesh cell strainer.Nail the spleen carefully to remove the big tissues. Avoiding big tissues influence the fusion of cells. Was the spleen cell suspension with RPMI 1640 medium.Then the cell suspension was added into the centrifuge tube. Harvest the spleen cells by centrifugation at 1, 000g for 10 minutes. And then discard the supernatant.The spleen cells were supposed to be at the amount of one billion to 10 billion. Remove the cultivated and amplified myeloma cells from the incubator and gently shake the flask to obtain a cell suspension. Was the suspension with RPMI twice.Mix the spleen cell suspension and the myeloma cells. Count the cells and adjust the concentration. The final ration of spleen cells to myeloma cells should be one to five to one to 10.Centrifuge the cell mixture and then remove the supernatant. Add one milliliter of 50%polyethylene glycol solution to the cells and gently stir at 37 degree for one minute. Standing for 30 seconds, add two milliliters of RPMI 1640 medium and incubate at 37 degree for two minutes to terminate the reaction.Add into two milliliters RPMI for another one minute. Then, add into 10 milliliters RPMI 1640. Centrifuge at 800g for 10 minutes.Discarding the supernatant, add HAT solution and continue to cultivate the cells for seven days at 37 degree 5%carbon dioxide. After seven days, cell supernatants in 96 well plates were aspirated after cell fusion and added into ELISA plates pre-coated with coating antigen. Cultivated at 37 degree for one hour, secondary antibody was added and cultivated for half an hour.After add 100 microliters of substrate solution. Stop the reaction. To the ELISA plates into the microplate reader, endpoint method was used read the data at 450 nanometer.Measured absorbance at 450 nanometer and screen the positive hybridomas. Use the limiting dilution method to prepare the the monoclonal hybridomas. After removing the HD medium from the selected hybridomas, re-suspend the cells in RPMI 1640 and count them.Dilute the cells of a concentration of one cell, two cells, and four cells per well by RPMI 1640 in a 96 well plate and culture. Transfer cells from the hybridomas that are positive to 24 well plates, 25 and 75 square centimeter flasks for cultivation. Store the myodomas in a gradient cooling box at minus 80 degree for 24 hours.Then transfer to liquid nitrogen for longterm storage. Titer of antiserums were determined by indirect ELISA. Mouse one through four was immunized with narigin BSA conjugate was significantly higher than the control mouse without immunized.The titer of one to 5, 000 is sufficient diffusion. The molecular weight of the hapten conjugate was confirmed by MALDI-TOF analysis. As shown in figure two, as a molecular weight of BSA standard and narigin have been known, we can calculate and determine that the molecules of narigin were conjugated with BSA.Only the hybridoma can survive and grow in HAT selection media. After seven days of growth, the cell culture can be tested by ELISA. The positive hybridomas were re-cloned and expanded.These images show the conventional outcome for stable monoclonal and polyclonal hybridoma cell lines. The critical point of this experiment is screening the mAbs specific for the hapten, but not carrier protein. The specificity of the mAb were then examined in the presence of other structurally related compounds.The cross reactivity was calculated to evaluate the mAb specificity to the antigen. The calibration curve of naringin and the liner range were obtained. The antigen specific monoclonal hybridomas were expanded and cryopreserved in liquid nitrogen for longterm storage.After watching this video, I hope you have a good understanding for how to generate monoclonal antibodies against a small moleculare compounds. So that's all. Thank you for your watching and good luck for your experiments.

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JoVE Journal

Immunology and Infection

10.7K Görüntüleme.  Beijing University of Chinese Medicine. Monoklonal antikorlar, tek bir B-lenfosit klonundan üretilen mono-spesifik antikorlar ve aynı epitopa bağlanan monovalent antikorlar olarak bilinir. Son zamanlarda, monoklonal antikor tabanlı ELISA gıdalar veya doğal ürünlerin nitel veya nicel analizi için gelişmekte olan platform haline gelmiştir. Bu yöntem sıkıcı ön işlem adımları olmadan doğru, hassas ve etkili bir yöntemdir.Bu biyolojik numuneler ve gelecekteki klinik uygulamalar için gereklidir. TCM mAD'lerin...