Background:

There are two custom peptides that act as target C.

Case design:

➢ Immunogen design & preparation

Target C, the two custom peptides, were conjugated to KLH as immunogen respectively (N-KLH-peptide and C-KLH-peptide).

➢ Immunization scheme design & conduction

We utilized a special water-soluble adjuvant in process of immunization to enhance immune response. Two CAMouse™ models were immunized 3-5 times at 2-week intervals. Meanwhile, another 5-10 CAMouse™ models of different strains/genetic backgrounds can serve as backup plans. For every single antigen, we guarantee our clients that good immune responses will be achieved (serum dilution at 1: 8000, ELISA OD450 > 1.0 or serum titer> 512,000).

The titering results after the booster immunization (as shown in Table 1.) revealed that the 2 mice both achieved highest titer of up to 1:1,000,000. We finally chose Mouse 1 for following monoclonal antibody development.

Table 1. Titering results after the booster immunization

[Green]: titer
a. 1st Ab: antisera
b. 2nd Ab: HRP-goat anti-mouse IgG (Fcr)

➢ B cell sorting by FACS

We can perform various protein labeling in vitro, such as fluorescent molecules or biotins. Here to facilitate B cell sorting, we linked FITC, one of the commonly used fluorescent molecule, to the antigen. After collecting peripheral blood of mouse #1, the antigen was conjugated and used as probes for sorting by fluorescence-activated cell sorting (FACS). It is not only highly accurate and efficient but also capable of high degree of automation.

➢ Specificity screening and validation

We provide multiple screening strategies depending on different target types/requirements. Since we had another peptide which can be used as positive control, we performed 4 times of ELISA screening to obtain B cell candidates with adequate affinity & specificity.

Figure 1 illustrated the results of 1^st - 4^th batch of supernatants ELISA test. 200 clones were picked and tested in total. It was shown that 116/126 clones could react with the control peptide and target C as well. Besides, there were 3 clones that could only bind with the control peptide but not with target C. Moreover, 6 clones could only bind with target C but not with the control peptide. A negative clone was also offered as the NC. All of these clones were expressed with IgG antibodies.

Figure 1 the results of 1st to 4th batch of supernatants ELISA test

➢ Antibody sequencing

To remove repeated clones, we first sequenced six top clones. Figure 2 showed the HC alignment of the six sequenced clones. Two unique clones were identified. The full-length antibody sequences can be revealed as fast as 2-3 weeks.

Figure 2 The HC alignment of the 6 sequenced clones

➢ Antibody production and purification

It’s necessary to obtain adequate number of antibodies with high purity for the downstream development (in vitro or in vivo). We have extensive experience in recombinant antibody expression (scFv/Fab/IgG) using a variety of systems. Both transient and stable transfection in a variety of featured host cell options are available. Of note, we also provide GMP-grade manufacture strain establishment service upon request.

Conclusions:

In this case, we developed a fully human customized antibody against target C by native B cell sorting technology. The patented CAMouse™ strain proves superior utility and efficiency in generating fully human antibody using B cell sorting technology.

Please note: we only produce antibodies for research purposes. Our products and services cannot be used directly on humans.