ELISA Development Services

SERVICE NO:BOTHYJ001

BOT Bioparticles is a comprehensive high-tech enterprise specializing in the research and development and production of high-precision, high-performance and high-value-added particle materials and proteins, providing high-precision biomedical materials and innovative diagnostic solutions. We can develop specific ELISA kits according to the individualized needs of customers. The kits can detect the content of specific components in the samples by methods such as ELISA sandwich method, competitive method (for determining antigens), indirect method (for determining antibodies), or competitive method (for determining small molecule compounds).  Paired antibodies refer to two antibodies that can bind to an antigen molecule simultaneously. BOT Bioparticles has a professional technical team and can screen out antibody pairs with high specificity and binding force for customers.

We have established a standardized reagent research and development and production base, enabling clinical testing to have high sensitivity and strong anti-interference performance. We focus on innovative development and provide full-cycle services for global customers. BOT Bioparticles has always adhered to the concept of diversified services, providing customers with diversified products and specialized services.

1. Immunogen Preparation

If you want to customize an antigen, please fill out this protein service inquiry form.

Protein Expression & Purification Service Quote

This form is aimed to know accurate info about clients and their requirements, so that we can provide faster quotation and better services. Please fill the form, we will reply to you within 1-2 business days upon receipt of this form.

Thanks for your cooperation.

Antibody Preparation

(1) Rabbit pAb generation

Introduction

Polyclonal antibodies are produced by multiple B lymphocyte clones, stimulated by various antigenic determinants and capable of binding to multiple antigenic epitopes. Rabbit polyclonal antibody is a rapid, economical and efficient method for preparing high-affinity antibodies. BOT Bioparticles has a professional antibody research, development and production team, and can provide customers with one-stop antibody preparation services including antigen design¡úpolypeptide synthesis, protein expression¡úanimal immunity¡úantibody purification¡úantibody labeling¡úimmunological detection and other projects. According to the specific experimental requirements of customers, we can provide polyclonal antibody preparation services for various animal species such as mice, rabbits, rats, chickens, and sheep. The prepared polyclonal antibodies can be used in various applications such as ELISA, Western Blot, IHC, IF, and IP.

Service Content

A. Customers can provide antigens for the preparation of polyclonal antibodies. The antigens include: natural proteins, recombinant proteins, cells or cell extracts, viruses or pathogens, bacterial strains or bacterial proteins, small molecule polypeptides, and compounds. Among them, small molecule polypeptides and compounds and other haptens need to be coupled with carrier proteins before immunization.

B. Customers can provide information such as protein name, GeneID, nucleic acid sequence, and amino acid sequence. Our company designs antigens based on the experimental requirements of customers, synthesizes polypeptide antigens or expresses protein antigens, and immunizes animals to prepare polyclonal antibodies.

Sample Requirements

1. The customer provides protein or polypeptide fragments required for antibody preparation, with antigen purity > 85%, quantity > 10mg, and concentration > 1mg/ml.

2. The antigen information provided by the customer should include the full English name of the protein antigen, species information, GeneID nucleic acid sequence or amino acid sequence information. Compound antigens should provide information such as chemical structural formula, active groups, and solubility.

Technical Process

1. Preparation of conventional polyclonal antibodies

2. Application in the preparation of polyclonal antibodies for specific experiments (such as WB, IHC, FCM, neutralizing antibodies, etc.)

The customer is required to provide the full English name of the target protein corresponding to the antibody preparation, species information, GeneID or sequence information

Note: The customer needs to provide the tissue, protein, cell and other samples required for the detection experiment in advance.

Service Description

1. Due to the diversity of immune antigens, for peptides that are difficult to synthesize, proteins that are difficult to express, or antigens with poor immunogenicity, both parties can negotiate to determine the preparation cycle and preparation cost.

2. Due to the fact that animal immunity is related to multiple factors, the polyclonal antibodies prepared have a certain degree of uncertainty. The conventional preparation of polyclonal antibodies is only responsible for the mutual binding between the obtained antibodies and the antigens used for the preparation of the antibodies, and is not responsible for other applications of the antibodies. If the customer needs to prepare antibodies for specific experiments, please choose the polyclonal antibody preparation BOTYW0002-3.

(2) Mouse mAb generation

Introduction

Monoclonal antibodies are produced by monoclonal hybridoma cells formed by the fusion of activated B lymphocytes with myeloma cells. Mouse monoclonal antibodies are common monoclonal antibodies. Meanwhile, mice are also one of the common model animals in the fields of biology and medicine.

Sample Requirements

The customer provides the protein or polypeptide fragments required for antibody preparation, with the antigen purity > 85%, quantity > 10 mg, and concentration > 1 mg/ml. If the antigen is a small molecule polypeptide, compound or other hapten, a carrier protein needs to be coupled before immunization. The types of carriers include BSA, KLH, HRP and OVA.

The antigen information provided by the customer should include the full English name of the protein antigen, species information, GeneID nucleic acid sequence or amino acid sequence information. Compound antigens should provide information such as chemical structural formula, active groups, and solubility.

Please explain the source of the sample, biological hazards and other special circumstances in advance.

Preparation of mouse monoclonal antibodies

BOT Bioparticles is capable of providing customers with mouse monoclonal antibody preparation services and preparing hybridoma cell lines using cell fusion technology. Hybridoma cells are formed by the fusion of myeloma cells and B lymphocytes that secrete antibodies (except for the fully human monoclonal antibody preparation technology), and they secrete monoclonal antibodies. Since they only act on a certain antigenic determinant on the antigen complex molecule, they have the characteristics of strong specificity and high titer.

A. Immunized animals

Immunizing animals is the process of immunizing mice with the target antigen to induce sensitized B lymphocytes in the mice. Healthy female Balb/c mice aged 6-8 weeks were selected and immunized according to the pre-specified immunization protocol. Antigens enter peripheral immune organs through the blood circulation or lymphatic circulation, stimulating the corresponding B lymphocyte clones, activating and proliferating them, and differentiating them into sensitized B lymphocytes. (Antiserum preparation - Immunizing animals)

B. Cell fusion, screening and subcloning

Mouse spleen cells with serum titers meeting the requirements were selected and fused with mouse myeloma cells. The fused cells were moderately diluted for cloning. Hybridoma cells were screened by HAT. Through immunogenogen detection, positive clones were selected for subclone expansion culture. Positive subclones were screened by ELISA and expanded culture was carried out. After fusion, one-time cloning by the limited dilution method and supernatant detection of mice, multiple cell lines that met the project requirements were obtained. Among them, the cell lines were selected for subsequent ascites preparation.

C. The large-scale preparation of monoclonal antibodies

(1) Induction method in animals

Balb/c mice were taken and pretreated by intraperitoneal injection of plantane or liquid paraffin. Intraperitoneal injection inoculation of hybridoma cells. When the abdomen of the mice expands. A large amount of monoclonal antibodies can be obtained by extracting ascites with a syringe.

(2) In vitro culture method

Hybridoma cells were cultured in large quantities in vitro using rotating culture tubes. Monoclonal antibodies can be obtained from the supernatant. By centrifuging and removing the cells and their debris, monoclonal antibodies can be obtained.

Purification of monoclonal antibodies

For ascites, the cells and their residues, small particulate matter, and fat droplets are removed by using silica adsorption and filtration centrifugation methods. For hybridoma cells cultured in vitro, monoclonal antibodies can be obtained from the supernatant. By centrifugation to remove the cells and their fragments, monoclonal antibodies can be obtained.

Antibody extraction: Silica adsorption method, ammonium sulfate precipitation method, caprylic acid-ammonium sulfate precipitation method, gel filtration, affinity chromatography, ion exchange chromatography and other methods were adopted.

Antibody identification: Identification was carried out by methods such as ELISA detection, SDS-PAGE, and Western Blot.

(3) Rabbit mAb generation

Introduction

Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have the outstanding advantages of high affinity, more diverse antigenic epitopes recognized, and the ability to recognize antigens that are not immunogenic in mice. This service project involves immunizing rabbits as per customer requirements, extracting B cells, constructing a phage display rabbit immune single-chain antibody library, and screening high-affinity rabbit single-chain antibodies.

Preparation of monoclonal antibodies in rabbits

Antigen preparation

Animal immunity

Cell fusion

Cell screening

Antibody purification

Delivery

Sample Requirements

The customer provides the protein or polypeptide fragments required for antibody preparation, with the antigen purity > 85%, quantity > 10 mg, and concentration > 1 mg/ml.

The antigen information provided by the customer should include the full English name of the protein antigen, species information, GeneID nucleic acid sequence or amino acid sequence information. Compound antigens should provide information such as chemical structural formula, active groups, and solubility.

3. ELISA Kit development

A. Antibody labeling

Introduction

Antibody labeling technology is a technique that labels some substances that are both easy to measure and highly sensitive onto specific antigen or antibody molecules. The enhanced amplification effect of these markers can reveal the nature and content of antigens or antibodies in the immune response system, and has been used in the analytical research and technical determination in fields such as medical pathology, immunohistochemistry, molecular biology, and biopharmaceuticals.

BOT Bioparticles can provide customers with small molecule/polypeptide/antibody/protein labeling services including biotin labeling, fluorescein labeling, enzyme labeling, etc.

Service content

1. Acceptable samples to be labeled

Small molecules/polypeptides/antibodies/proteins

2. Markers that can provide marking services

Enzyme-labeled substances: AP/ALP, HRP;

Biotin;

Conventional fluorescein: FITC, Rhodamine B;

Cy series fluorescein: Cy3, Cy5, Cy5.5, Cy7;

Alexa Fluor series fluorescein: AF350, AF488, AF555, AF594, AF647;

Fluorescent proteins: Phycocyanin (PE), phycocyanin (APC);

Fluorescence double labeling: PE-Cy3, PE-Cy5, PE-Cy5.5, PE-Cy7;

Colloidal gold is labeled Co-Au

Sample Requirements

Purity >90%

Specification >1mg

Concentration >1mg/ml

Antibody: Free of BSA, sodium azide, glycine, etc

Protein: Information such as molecular weight, isoelectric point, buffer solution composition and pH should be provided

Polypeptide: Provides amino acid sequence

Small molecule compounds: Provide molecular structural formulas

Note: For other marking services, please contact us for customization

B. Antibody matched pair screening

Introduction

Paired antibodies refer to two antibodies that can bind to an antigen molecule simultaneously. Under normal circumstances, an antigen molecule has multiple antigenic determinants. When an antigen is injected into an animal for immunization, different antibodies will be produced against different antigenic determinants. The antibodies produced have specificity and exclusivity, that is, an antibody molecule will only specifically bind to one antigenic determinant. Two antibodies targeting different antigenic determinants may simultaneously bind to antigen molecules. If two antibodies bind to the same antigen molecule simultaneously, then these two antibodies are paired antibodies. Paired antibodies are generally applied in double-antibody sandwich ELISA experiments.

BOT Bioparticles can develop double-antibody sandwich antibody pairings suitable for the specific application requirements of customers according to their demands, enabling them to be applied in fields such as ELISA kit development and colloidal gold test strip development.

Service content

Recombinant protein expression

Antibody preparation

Antibody purification and labeling

Antibody pair screening

Data analysis

Delivery result

1. Antigen: 1-3 mg

2. Multi-antibiotic products

3. Monoclonal antibody products

4. Antibody pairs: Low-assurance pair (mAb, mAb or mAb, pAb); If there are additional pairings, provide them simultaneously;

5. Paired antibody labeling

6. ELISA kit

7. Test reports and data

C. Assay development and optimization

D. ELISA Kit manufacturing

Delivery result

ELISA Kit

Experimental Report

Ensure a high success rate and a fast delivery speed;

Abundant antibody expression vector resources, strict quality control system and GMP-level operation level ensure reliable results;

High antibody titer: Prepare guinea pig monoclonal antibodies with high titer and high sensitivity to ensure delivery volume and meet experimental requirements;

Service-oriented: Reasonable prices, and comprehensive analysis based on customer needs can be conducted to provide satisfactory results for customers;

Professional technical support ensures strong, considerate and timely pre-sale and after-sale service support, and is always ready to answer any questions you may have;

Complete upstream and downstream services: We offer one-stop technical services covering antibody preparation, antibody sequencing, and antibody labeling and modification, saving customers' precious time and funds.

Please contact our customer service department for more information or to discuss your procurement requirements.