Generation of Monoclonal Antibodies Against Natural Products

Summary

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.

Abstract

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 (<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.

Introduction

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 cancer^2,3. The study of natural products has made rapid progress, largely due to t....

Protocol

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 (<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 labeled a hapten. Hapten conjugation is a necessary and effective strategy for mAb production. To avoid cross reactivity, two different protein carrie....

Results

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 .......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
800 mesh (40 μm nylon) filter	FALCON	352340
24 well culture plate	NUNC	119567
25 cm2 Flask	Labserv	310109016
3,3’,5,5’-Tetramethylbenzidine(TMB)	Sigma Aldrich	860336 1G
75 cm2 Flask	Corning	430720
96 well culture plate	NUNC	117246
bovine serum albumin	AMRESCO	332
cell strainer	FALCON	352340
centrifuge tube 15 mL	Corning	430645
centrifuge tube 50 mL	Corning	430828
cryotubes, 1 mL	Sigma Aldrich	V7384-1CS
cultivator	DRP-9082	Samsung
dialysis membrane (10kDa)	Heng Hui	45-10000D
dimethylsulfoxide	Sinopharm Chemical	DH105-10
electronic balance	BS124-S	Sartorius
ELISA plates, 96 well	NUNC	655101
ethanol, 96%	Sinopharm Chemical
Fetal bovine serum	Gibco	16000-044
fetal calf serum	Invitrogen	10270106
Freund´s adjuvant, complete	Sigma Aldrich	SLBM2183V
Freund´s adjuvant, incomplete	Sigma Aldrich	SLBL0210V
Gelatin	AMRESCO	9764-500g
Gradient cooler container	Nalgene	5100-0001
HAT media supplement	Sigma Aldrich	H0262-10VL
HRP-conjugated goat-anti-mouse IgG antibody	applygen	C1308
HT media supplement	Sigma Aldrich	H0137-10VL
Inverted Microscope	IX73	Olympus
keyhole limpet hemocyanin	Sigma Aldrich	H8283
MALDI-TOF-MS	Axima-CFR  plus	Axima
Microplate Reader	BioTex	ELX-800
mouse	Vital River	BALB/c
ovalbumin	Beijing BIODEE	5008-25g
PEG	Sigma Aldrich	RNBC6325
Penicillin&Streptomycin solution	Hyclone	SV30010
Pipette 10 mL	COSTAR	4488
Pipette 25 mL	FALCON	357525
RPMI 1640	Corning	10-040-CVR
skim milk	applygen	P1622
sodium periodate	Sinopharm Chemical	BW-G0008
Sulfo-GMBS	Perbio Science Germany	22324
TipOne Tips 1,000 µL	Starlab	S1111-2021