Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

The overall goal of this procedure is to clone and express antibodies produced by B cells. This is accomplished by first isolating B cells from a relevant source tissue like blood or from thymus. The second step is to perform facts and select the B-cell population positive for IgG production.

Next, the cells are immortalized by infection with Epstein-Barr virus together with toll-like receptor nine activation by C-P-G-O-D-N 2006 and kept in culture. The final steps are to isolate RNA from clones that are tested positive for IgG production by Eliza and to generate copy DNA from both light and heavy IgG chains. Ultimately, the IgG sequences can be cloned in mammalian expression vectors to produce high quantities of monoclonal human IgG.

The main advantage of this technique over existing methods like phage display technology is that original IgG heavy and light chain pairing is not lost. This method can answer key questions in the field of autoimmunity such as discovery and functional characterization of pathogenic autoantibodies. Don't forget that working with patient material and EBV virus can be risky.

EB virus is a human pathogen and precautions such as VSL two PROEs and wearing adequate protection should always be taken while performing these steps Before staining the P BMCs, plate them in a 25 centimeter flask with six milliliters of complete RPMI 1640 medium and let them recover overnight. After blocking and washing the cells, collect the pbmc in a tube with a half milliliter of medium and spin them down. Then resuspend the cells in labeling buffer and count them.

Next, prepare two fax tubes of 5 million cells in 200 microliters of buffer to these tubes. Add 10 microliters of anti CD 22 P-E-R-C-P or 40 microliters of anti IgG pe and let them incubate for 30 minutes on ice and in the dark, then wash, filter, and sort the cells. The goal is to count the double positive cells.

Begin with diluting the sorted P BMCs one cell per microliter in RPMI 1640, complete medium with addition of C PG 2006 and EBV. Then in a hood seed 110 microliters of the cell dilution with EBV and CPG 2006, two wells of a 96 well round bottom plate previously plated with WI 38 irradiated cells. The infection of 50 cells per well is optimized and increases the likelihood to obtain a monoclonal antibody.

However, we recommend to confirm this by A PCR. After a week of incubation, inspect the cells under a microscope for B-cell growth. After two weeks, change the media slowly aspirate 90 microliters of medium from each well and transfer it to a new plate.

To test for IgG production by Eliza. Then add back 100 microliters of fresh supplemented medium, including IL two to each. Well continue monitoring for clone growth through their third and fourth week of growth.

Repeating the media change each week. See the text protocol for clonal expansion methods. Check the super named from the medium changes for IgG production using an enzyme linked immunosorbent assay or Eliza.

First, prepare the ELIZA plates to each well dispense 50 microliters of goat anti-human FC antibodies, diluted one to 200 encoding buffer. After incubating and washing the plate, block the wells with 100 microliters of blocking buffer per well and incubate the plate again. After the block incubation, knock the liquid out of the plate onto a paper towel.

With the plate prepared. Dilute the supernatants with four volumes of RPMI. Then for standard controls, make a serial dilution of human IgG solution in RPMI from 1000 nanograms per milliliter to 15.6 nanograms per milliliter.

Also include a blank. Apply 50 microliters of the supernatant or standard to each well and incubate the ELIZA plate for an hour at 37 degrees Celsius in the same manner, additional Eliza S can be set up to analyze further dilutions of the supernatants to test the antibody affinity. After the incubation wash the plate.

Then to each well apply 50 microliters of incubation buffer with peroxidase conjugated fab two. After another hour of incubation and another wash, add 100 microliters of substrate solution with TMB to each. Well then incubate the plate up to 10 minutes until the blue that appears stabilizes.

Then stop the reaction and measure the absorption. Any clone supernatants with measured absorbance. Three standard deviations over the blank are positive.

For human IgG antibodies, antigenic specificity can be detected by further assays. To begin, follow the text protocol to synthesize copy DNA from the positive clones. Then set up PCRs for the IgG heavy kappa and lambda chain.

In the results, find the heavy chain at 400 to 550 base pairs. Find the light chain at 300 to 400 base pairs. If they aren't there, use a microliter of products as a template for another PCR.

The new products should be in the appropriate range. Then proceed with cloning the best products First, sequence them. Their electropherograms should be clean and correspond to a single immunoglobin sequence.

Following the text protocol. Clone the purified PCR product into a microgram of plasmid DNA. Be sure to ligate overnight at 16 degrees Celsius.

Using T four DNA Ligase, prepare the HX cell in supplemented DMEM and grow them to 75%co fluency in 75 square centimeter culture flasks. For each heavy light chain transfection, prepare a mix with 1.75 Milliliters of DMEM without serum include 4.5 micrograms of heavy chain vector, three micrograms of light chain vector, and 50 microliters of polyethaline solution. Gently add this mixture to a plate of HX cells and swirl the solution over the plate.

Incubate the plate overnight and then begin collecting the culture media every day. Replacing it with serum less medium. The media contains the antibodies and it can be collected daily for four days after staining CD 22 and IgG positive p BMCs were sorted by facts.

The area of the double positive cells, B cells producing IgG antibodies was collected separately for expansion. About 1%of the pbmc were in this double positive population. After five weeks of EBV immortal and CPG 2006 stimuli, immortalized clones were isolated.

WI 30 feeder cells have a more elongated fibroblast shape, and the B cell clones appear as very small round cells growing clustered in the middle of the round bottom multi-well plate. The supernatant of the growing clones was tested using an ELIZA to IgG. A positive clone was evaluated on one with an eli, a value three standard deviations over the blank value.

At least three supernat from a positive clone tested positive in the eli. A antibodies from a positive clone were sequenced after cloning the immunoglobulin heavy and lambda chain pair variable and constant regions of the heavy and light chain could be characterized. Following this procedure.

Other methods like sequencing can be performed in order to answer additional questions like antibody, gene usage, genetic diversity, et cetera. After this development, this technique paved the way for researchers in the field of autoimmunity to explore disease, pathology, and relevant therapeutic strategies for autoimmune diseases like myasthenia gravis. The implications of this technique extend toward diagnosis and therapy of diseases like myasthenia gravis because discovery of pathogen antibodies can lead to the development of new treatments such as competitor antibodies, lacking inflammatory activity.