The MARK & MARL references have been SYnAbs' best sellers for many years.

The MARK monoclonal - mouse anti-rat kappa light chain - and MARL - mouse anti-rat lambda light chain - have more than one trick up their sleeve. But only those who already use them know it.

For all those who do not know us, or who are still hesitating to test SYnAbs products, let's discover some examples of research conducted with these practical and efficient tools!

And for those who already use them, a reminder never hurts, right?

In 1994, Gmür et al (1) wanted to investigate the habitat of periodontal pathogens. Analysis a battery of Gram-negative bacteria such as Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Porphyromonas gingivalis and Prevotella intermedia, they decided to use a sensitive quantitative immunofluorescence assay for detection. Binding of the rat mAbs specific for bacteria strains was visualized by a sandwich assay with SYnAbs MARK-1 reference, which is directed against the Kappa light chain of rat immunoglobulin.

Generated results suggested that the investigated periodontal bacteria are not "exogenous pathogens", but amphibiotic, opportunistic microorganisms which may have a natural habitat in the supragingival plaque of the interproximal area of molars.

In 2006, Cousins et al. (2) looked at the genetic causes of autoimmune diseases. They decided to bred PVG-RT1u, lymphopenia (lyp)/lyp rats, congenic for the iddm1 (RT1u) and iddm2 (lyp, Gimap5−/−) and to compare them to wild type congeners.

Using SYnAbs mouse anti-rat IgG1 (MARG1-2), mouse anti-rat IgG2b (MARG2b-2), mouse anti-rat IgE (MARE-1) and mouse rat anti-Igκ+λ (MARK-1/MARL-15), they investigate autoantibodies content in serum. Antibody levels were determined by comparison with SYnAbs isotype controls rat IgG1 (IR27), rat IgG2b (IR863), and rat IgE (IR162).

Curiously enough, Cousins et al. were witnesses of the production of Ig autoantibodies in lyp/lyp rats but the absence of IgE, which may reflect a phenomenon of absorption by tissue components.

In 1994, at the University of Louvain, Bombil et al. (3) were investigating the human B lymphoproliferative syndrome (huBLPS) following the transfer of human peripheral blood mononuclear cells (hu-PBMC). They generated LO-CD2a, a rat monoclonal antibody anti-human CD2, later rebranded MEDI-507 and today known as Siplizumab, able to inhibit in vitro T cell activation.

Working on hu-SCID and CB-17 SCID mouse models, the team got the opportunity to successfully use a large set of SYnAbs monoclonal antibodies to monitor both LO-CD2 (using MARK-1 as Anti-Drug Antibody) and animal immune response: MARK-1 mouse IgG1 anti-rat kappa light chain, MARG2b3 mouse IgGD anti-rat IgG2a mAb, LO-CD4b rat IgG2a anti-human CD4, LO-MOI rat IgG2b anti-human CD14, LO-PanB-a rat IgG2b anti-human CDW78, LO-HK-3 rat IgGDc anti-human kappa light chain, LO-HL-2 rat IgG1 anti-human lambda light chain, LO-HM2 rat IgGDc anti-human mu heavy chain, LO-HG-22 rat IgG2c anti-human IgG, LO-MK-1 rat IgG2a anti-mouse IgG; LO-MK-2 Rat IgG2a anti-mouse kappa light chain, IR418 rat IgG2a isotype control and IR863 rat IgG2b isotype control.

The experiments reported how crucial was the presence of functional T lymphocytes for a graft to take and for development of huBLPS in hu-PBMC-reconstituted SCID mice, since inhibition of T lymphocyte by a rat anti-(human CD2) monoclonal antibody (LO-CD2a) during the first 10 days of the graft prevented successful engraftment of human normal lymphocytes as well as hu-BLPS in SCID mice.

In 1995, Lepault et al (4) were working on diabetes. The leakage of blood cells to the islets of Langerhans seemed to depend on the expression of a set of molecules, most of which remained to be defined. One of this molecule, L-selectin, seemed of interest. In order to evaluate further the role of L-selectin expression on the development of autoimmune diabetes, the team decided to study the effect of Mel-14 antibody (rat IgG2a anti-L-selectin) treatment at different stages of the disease process in NOD mice in vivo.

Using SYnAbs IR418 as isotype control rat IgG2a, and MARK-1 (SYnAbs mouse anti-rat kappa light chain) to reveal Mel-14 as a second step reagent, Lepault et al demonstrated that diabetogenic cells in adult mice share phenotypic characteristics with activated/memory cells, and enter the pancreas using L-selectin-independent migratory pathways.

In 2008, Daley et al. (5) were investigating transplantation tolerance. In fact, in non-human primates, treatment with antibodies specific for the costimulatory molecule CD40L has proven an effective means to enable the prolonged acceptance of organ transplants. Nevertheless, unexpected occurrence of thromboembolism resulted in the abrupt termination of Tox and clinical trials. Consequently, the team developed a rat Fc-disabled anti-CD40L antibody.

Revealed with SYnAbs MARK-1/MARL15, this therapeutic candidate showed that short pulses of anti-CD40L antibody therapy might still be useful in tolerance protocols even when the Fc region is disabled.

Also working on the CD40L, Sandin et al. (6) wanted to investigate whether local low-dose agonistic of rat IgG2a anti-mouse CD40 antibody injection could be as efficient as an equivalent dose delivered systemically, and to establish whether the alternative administration route could reduce toxicity.

Quantification of circulating anti-CD40L antibody was performed by ELISA using SYnAbs anti-rat IgG2a (MARG2a-1) and SYnabs mouse-α-rat κ/λ light chain (MARK-1/MARL-15). Results stated that local low-dose anti-CD40L therapy is superior to systemic treatment and reduces circulating antibodies.

Approximately 95% of rat immunoglobulin light chains are of the kappa type. The allotypy in the rat species is located on the constant part of the kappa light chain.

To purify rat antibodies, protein A is not efficient. Protein G captures IgG2a isotype with a medium results, and IgG1 with poor outcomes. Protein L can bind IgG1, IgG2a, IgG2b, IgG2c, IgM, but through variable region kappa light chain.

By use of a MARK mouse monoclonal antibody with specific binding affinity for the Igκ-1a allotype on the kappa light chains of the LOU inbred rat strain, it is possible with immunoaffinity chromatography to isolate 95% (Igκ-1a-bearing immunoglobulins, including the immunoglobulins, of rats of Igκ-1b allotype) of all rat antibodies without cross-binding to bovine immunoglobulin of serum.

REFERENCES:

1. Gmür R, Guggenheim B. Interdental supragingival plaque, a natural habitat of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, and Prevotella nigrescens. J Dent Res. 1994 Aug;73(8):1421-8. doi: 10.1177/00220345940730080501. PMID: 8083438.
2. Cousins L., Graham M., Tooze R., Carter C., Eosinophilic bowel disease controlled by the BB rat-derived lymphopenia/Gimap5 gene. Gastroenterology 2006, BASIC–ALIMENTARY TRACT VOLUME 131, ISSUE 5, P1475-1485.
3. Fataki Bombil; Jean Pierre Kints; Xavier Havaux; Jean Marie Scheiff; Hervé Bazin; Dominique Latinne (1995). A rat monoclonal anti-(human CD2) andL-leucine methyl ester impacts on human/SCID mouse graft and B lymphoproliferative syndrome. , 40(6), 383–389. doi:10.1007/bf01525389 
4. Lepault F, Gagnerault MC, Faveeuw C, Bazin H, Boitard C. Lack of L-selectin expression by cells transferring diabetes in NOD mice: insights into the mechanisms involved in diabetes prevention by Mel-14 antibody treatment. Eur J Immunol. 1995 Jun;25(6):1502-7. doi: 10.1002/eji.1830250605. PMID: 7542194.
5. Daley SR, Cobbold SP, Waldmann H. Fc-disabled anti-mouse CD40L antibodies retain efficacy in promoting transplantation tolerance. Am J Transplant. 2008 Nov;8(11):2265-71. doi: 10.1111/j.1600-6143.2008.02382.x. Epub 2008 Sep 8. PMID: 18782294.
6. Linda C. Sandin, Anna Orlova, Erika Gustafsson, Peter Ellmark, Vladimir Tolmachev, Thomas H. Tötterman, Sara M. Mangsbo; Locally Delivered CD40 Agonist Antibody Accumulates in Secondary Lymphoid Organs and Eradicates Experimental Disseminated Bladder Cancer. Cancer Immunol Res 1 January 2014; 2 (1): 80–90. https://doi.org/10.1158/2326-6066.CIR-13-0067