C-reactive Protein (CRP)

Description

CRP was first identified in 1930 as a substance in the serum of patients with acute inflammation that reacted with the "C" carbohydrate antibody of the capsule of pneumococcus (Tillett and Francis 1930). CRP is a pentameric protein synthesized by the liver and is an acute-phase reactant protein that is primarily induced by the IL-6 action on the gene responsible for transcription of CRP during the acute phase of an inflammatory/infectious process. There are numerous causes of an elevated CRP, including acute and chronic conditions, and these can be infectious or non-infectious in etiology; trauma can also cause elevations in CRP (alarmin response), and more modest elevations tend to be associated with a broader spectrum of etiologies, ranging from sleep disturbances and aging to periodontal disease (Nehring and Patel 2019). There are no available assays for marmoset CRP.


Alignment

Protein alignment for human, rhesus macaque and marmoset CRP:

image of Protein alignment for human, rhesus macaque and marmoset C-reactive Protein (CRP)

 

Protein alignment for marmoset, owl monkey, and squirrel monkey CRP:

Protein alignment for marmoset, owl monkey, and squirrel monkey CRP

 

References

  • Nehring, S. M. and B. C. Patel (2019). C Reactive Protein (CRP). StatPearls. Treasure Island (FL).
  • Tillett, W. S. and T. Francis (1930). "Serological Reactions in Pneumonia with a Non-Protein Somatic Fraction of Pneumococcus." J Exp Med 52(4): 561-571.

 

Status

Anti-marmoset C-reactive protein (MAR CRP) monoclonal antibodies (mAbs) were generated by immunizing mice with several recombinant molecules, including MAR CRP with a C-tag (MAR CRP.C-tag), or C-tag plus immunogenicity tag. A total of 16 clones were selected after screening using ELISA, FluoroSpot (for clonality analysis) and Octet (for affinity scouting and epitope binning). Clones were amplified in small scale, and pure and biotinylated forms of each mAb were produced for testing against MAR samples in an ELISA format that tested all possible capture/detection combinations. An example of the outcome of the ELISA test is shown in Figure 1.

NWMIR-ELISA screening of capture/detection mAb pairs specific for MAR CRP
Figure 1. ELISA screening of capture/detection mAb pairs specific for MAR CRP

The result of the screening ELISAs indicated that many mAb combinations were able to recognize the recombinant MAR CRP molecule, as well as the native MAR CRP molecule present in the plasma of marmosets challenged with LPS. Soma mAb combinations also recognize CRP in the plasma of rhesus macaque after exposure to components of bacterial walls. Interestingly, only a small number of mAb combinations recognized a human CRP standard.

NWMIR-ELISA assay for MAR CRP to determine crossreactivity with other nonhuman primates and proper dilution of plasma samples
Figure 2. ELISA assay for MAR CRP to determine crossreactivity with other nonhuman primates and proper dilution of plasma samples.

One of the mAb antibody pairs that detected the human and marmoset CRP standards, and CRP in the plasma of marmoset and rhesus macaques, was the BZ22 and BZ24 pair. Thus, a new ELISA assay was using each antibody as capture or detection, and testing samples from the other NWM. We also prepared dilutions of plasma to determine the sensitivity of each antibody pair. Figure 2 shows that the BZ22 capture/BZ24 detection combination produced the highest signal for NWM samples, and detected CRP in plasma of rhesus macaque. The BZ22 capture/BZ24 detection was also the most sensitive combination for the detection of recombinant human and marmoset CRP (Figure 3).

NWMIR-ELISA assay for MAR CRP.
Figure 3. ELISA assay for MAR CRP. The BZ22/BZ24 mAb pair were used in different order to identify the sensitivity of detection of human or marmoset recombinant CRP, starting at 20 ng/ml.

In summary, we have generated mAbs that bind native MAR CRP and identified mAbs pairs that work in ELISA format for marmoset, squirrel monkey, owl monkey, and rhesus macaque CRP. The BZ22/BZ24 mAb pair will be tested on the LMX platform.