LO-MM is one of the key references in the SYnAbs antibody catalogue, but in reality very few people know exactly what this antibody has been used for in the past and the potential of the LO-MM range through LO-MM-3, LO-MM-8 and LO-MM-9, antibodies of different affinity and isotype.

LO-MM means firstly that it is an antibody generated through the immunization of SYnAbs proprietary species rat-LOU (Louvain-la-Neuve, Belgium), electrofusion with myeloma rat-LOU IR983F, and directed against the mouse IgM antigen.

In fact, the first letters refer to the species, and then to the targeted antigen. In this case, the M in LO-MM refers to the mouse species, and the second letter M to the IgM isotype.

• LO-MM-3 is a IgM kappa rat anti-mouse heavy chain
• LO-MM-8 is a IgG1 kappa rat anti-mouse heavy chain
• LO-MM-9 is a IgG2a kappa rat anti-mouse heavy chain

Any LO-MM reference is specific for the mouse species, and doesn’t bind to human, chicken, rabbit, goat, sheep, bovine, horse, dog, swine, or baboon immunoglobulins.

Now let's find out what this secondary monoclonal antibody has been used for.

The most common and obvious application for the use of a secondary antibody is the detection and quantification of the antigen, in this case mouse IgM. Mouse IgM is secreted non-specifically during the primary immune response, following the presence of a non-self element, and is then replaced by a specific IgG-type response.

For instance, Huang et al. has developed an ELISA used to quantify the amounts of IgM in mouse serum with capture mAbs anti-mouse IgM LO-MM-3 and detection mAbs biotin-conjugated anti-mouse IgM LO-MM-9, to monitore immune isotypic response to specific dust mite allergen (1). Another study we can mention is the one of Savignac et al (2) where the team studied the B cell proliferation of specific transgenic mouse model compared to wild-type strain by measuring levels of circulating Ig isotypes.

But besides characterization and quantification of IgM, a less widespread application of the LO-MM antibody is immunoaffinity chromatography for the purification of mouse IgM, as performed by Cormont et al (3).

There are currently several methods of purifying IgM.

In the past, it has been determined that the best purification schemes are ion exchange chromatography, a gel filtration column, in conjunction with precipitation, reduction and alkylation, i.e. numerous steps with a fairly low final yield.

To improve this yield and reduce the number of steps in the laboratory, it is possible to use kits using, for example, mannan-binding protein (MBP). MBP is a mannose-specific lectin, but purification of IgM from species other than human and mouse serum has not yet been optimised. In addition, IgM purification with this system is temperature dependent and the binding and washing steps must be performed at 4°C, which is not always practical. In terms of cycling, immobilised MBP can usually be regenerated at least 10 times with no apparent loss of binding capacity.

To overcome these problems, at SYnAbs we use rat monoclonal antibodies against mouse IgM to obtain a percentage of purified IgM recovered in excess of 90% in a single step. LO-MM grafted columns can be reused up to 100 times.

While LO-MM is capable of detecting circulating antibodies, this secondary antibody reference is also capable of targeting membrane IgM on the surface of B lymphocytes. This type of property leads to different analyses and very interesting publications on the role of different B populations.

This is typically the case for the work of Denis et al (4).

In the 1990, it was generally accepted that B cells could act as antigen presenting cells (APCs) in vitro, but their relative role in vivo remained controversial. In this study, Denis et al. used LO-MM rat mAbs to target in vivo or in vitro antigen to membrane IgM on B cells and analyse the induction of the humoral response.

Results demonstrated that injection of BALB/c mice with LO-MM-9 rat mAb directed against IgM strongly enhances the IgG1 antibody response Moreover, purified resting B cells were shown to induce a specific IgG1 response in vivo only when they were cultured with rat mAb against mIgM or mIgD but not with myeloma rat Ig of the same isotype.

B cells do not need to be activated to present antigen since the induction of the specific antibody response does not correlate with the mitogenic activity of rat mAb nor with the IgG1 polyclonal synthesis in vivo. These data clearly show that resting B cells can present antigen in vivo and induce an antibody response, and underline the importance of membrane IgM as targets for antigens.

Indeed, previous data has shown that the specific humoral response induced by the injection of antigen-pulsed purified dendritic cells was characterized by the secretion of lgG2a antibodies. Therefore, the absence of specific lgG2a in Denis’ system strongly suggested that B cells, not dendritic cells, were the in vivo APCs.

But SYnAbs' LO-MM references are much more than binder antibodies for their target.

In fact, our background in the Experimental Immunology Unit (Faculty of Medicine, University of Louvain, Brussels, Belgium) has enabled us to develop effector antibodies that have led to our international recognition in the generation of depleting therapeutic antibodies such as:

• Siplizumab (anti-CD2, licensed to BioTransplant and then AstraZeneca),
• LO-TACT (anti-CD25),
• Syn-CCR8 (anti-CCR8)
• and above all...(yes, you get it) LO-MM!

To overcome the shortage of organ donors in allotransplantation, discordant xenografts from pigs to humans were seen as a potential solution. However, the success of this approach is limited by hyperacute rejection, mainly resulting from the binding of recipient XNA to the donor endothelial cells. The XNA are principally of the IgM isotype and directed against the α-(1,3) galactosyl epitope.

A number of strategies have been investigated in developing effective methods to eliminate XNA before transplantation, such as immunoadsorption, plasmapheresis and anti-μ monoclonal antibody (mAb) injection, developed by Soares et al. (5) in our laboratory, that induce circulating IgM depletion but only an incomplete B-cell suppression.

These measures have been partially effective in preventing hyperacute rejection of xenogeneic organs. B-cell depletion, particularly of those cells responsible for XNA production, is seen as a potential alternative to achieve not only the elimination of circulating XNA but also the induction of tolerance in newly formed B cells after the xenograft contact.

Chentoufi and Nizet (6) were the first to demonstrate that in adult mice treatment with SYnAbs LO-MM anti-μ induces a complete depletion of B cells and xenoreactive natural antibodies (XNA) and represents a potential approach to induce xenograft tolerance.

In 1994, Denis et al. (7) demonstrated that a total depletion of circulating IgM was first observed in adult mice using a single dose of 40 mg/kg of a rat anti-mouse IgM mAb (LO-MM-9) without serum sickness or glomerulonephritis. Similarly, administration of 1 mg of rat anti-mouse IgM mAb (LO-MM-9) was also capable of internalizing membrane IgM on B cells for 5 days.

Since LO-MM can induce B cell and secreting IgM depletion as previously described, it’s possible to study the spread of an infection in the body and the associated immune response in a context of immunosuppression.

This is the case of the Listeria monocytogenes study conducted by Cerny et al (8).

The role of immunoglobulin in immune responses to infectious agents varies with the model infection studied. Whereas natural resistance to facultative intracellular bacteria depend upon take and initial inactivation by phagocytes, subsequent T-cell-mediated protection is due mainly to activation of macrophages to become efficiently bactericidal. Both T-cell-dependent macrophage activation and the presence of specific antibodies seem to be required for protection against Salmonella typhimurium but the relative roles were unclear for immunity to Mycobacterium tuberculosis or for Listeria monocytogenes.

Affinity-purified LO-MM-9 was used at a concentration of 2 mg/ml. To initiate B-cell suppression, the team treated neonatal mice with 0.1 ml of LO-MM-9 intraperitoneally on days 1, 2, 3, 5, 7 and 9. For maintenance of suppression, mice were injected once a week with 0.3 ml IRS or 600μg of LO-MM-9. Offspring of females treated with anti-IgM were treated similarly; these mice were not only B-cell-depleted but also agammaglobulinaemic. Control mice were kept under identical conditions and were either injected with normal rabbit sera (NRS) or chromatographically purified rabbit IgG (NRP).

B-cell- and antibody-depleted mice showed an unimpaired, efficient anti-Listeria immunity that was comparable to that in normal mice, if immunosuppression was achieved by treatment of BALB/c mice with LO-MM-9 monoclonal rat anti-mouse IgM antibody. The fact that the rat anti-mouse IgM treatment of mice born and raised by similarly immunosuppressed mice show an anti-Listeria immunity that is identical to that of untreated BALB/c, certainly proves formally that neither "natural" pre-existing antibodies nor specifically induced ones play a significant role in primary anti-Listeria immunity assessed in vivo directly in the mouse.

If we have to draw a mirror image in infectiology, we can mention the study on the LCMV virus conducted by the same team (9).

Applying a similar protocol to the one described previously for Listeria monocytogenes study, Cerny proved that LCMV reached higher titers and persisted for a longer time in immunosuppressed mice. However, the mice were able to eventually clear the virus successfully, but clearance was delayed compared with that for B-cell-competent mice.

The results suggested some role of antibody in the control of LCMV growth, spreading, or both but indicate also that antibodies, although helpful, are not mandatory for complete LCMV elimination. Overall, these experiments with immunosuppressed and with agammaglobulinemic mice demonstrated that T cells alone might eventually clear LCMV completely but that antibodies, although not mandatory, might accelerate virus clearance significantly.

REFERENCES

(1) Airway inflammation and IgE production induced by dust mite allergen-specific memory/effector Th2 cell line can be effectively attenuated by IL-35 (In The Journal of Immunology on 1 July 2011 by Huang, C. H., Loo, E. X., et al.)

(2) Increased B cell proliferation and reduced Ig production in DREAM transgenic mice (In The Journal of Immunology on 15 December 2010 by Savignac, M., Mellstrom, B., et al.)

(3) Cormont F., Manouvriez P., De Clercq L., Bazin H. The use of rat monoclonal antibodies to characterize, quantify and purify polyclonal or monoclonal mouse IgM. Meth. in Enzymol. 1986,121:622-631

(4) Denis O, Latinne D, Nisol F, Bazin H. Resting B cells can act as antigen presenting cells in vivo and induce antibody responses. Int Immunol. 1993 Jan;5(1):71-8. doi: 10.1093/intimm/5.1.71. PMID: 8443123.

(5) Depletion of IgM xenoreactive natural antibodies by injection of anti-mu monoclonal antibodies. Dominique Latinne; Miguel Soares; Xavier Havaux; Frangoise Cor Mont; Beth Lesnikoski; Fritz H. Bach; Herve Bazin (1994). Depletion of IgM Xenoreactive Natural Antibodies by Injection of anti-μ Monoclonal Antibodies. , 141(1), 95–125. doi:10.1111/j.1600-065x.1994.tb00874.x

(6) Chentoufi AA, Nizet Y, Havaux X, De La Parra B, Cormont F, Hermans D, Bazin H, Latinne D. Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells. Transplantation. 1999 Dec 15;68(11):1728-36. doi: 10.1097/00007890-199912150-00017. PMID: 10609950.

(7) In Vivo Study of mIgM and mIgD CrossLinking on Murine B Cells. Scand J Immunol 1994;39:625-32. Denis O, Macedo-Soares F, Latinne D, Nisol F, Bazin H. https://www.sciensano.be/sites/default/files/denis_et_al-1994 scandinavian_journal_of_immunology.pdf

(8) Anti-Listeria monocytogenes immunity in/z-suppressed mice: a comparison of treatment with conventional hyperimmune rabbit anti-mouse IgM and affinity-purified, monoclonal rat anti-mouse IgM. A. Cerny , A. W. Hügin, H. Bazin, S. Sutter, H. Hengartner, and R. M. Zinkernagel.

(9) Clearance of Lymphocytic Choriomeningitis Virus in Antibody- and B-Cell-Deprived Mice A. CERNY, S. SUTTER,' H. BAZIN, H. HENGARTNER, AND R. M. ZINKERNAGEL Institute of Pathology, University Hospital, CH-8091 Zurich, Switzerland, and Experimental Immunology Unit, University of Louvain, Brussels, Belgium