Table 1.
Selected examples of the use of nanobodies (and V-NAR) in disease diagnosis
| Antigen (target protein or small molecule) | Application | Method | Reference |
|---|---|---|---|
| Detection of parasite or fungal infection | |||
| Malarial apical membrane antigen-1 | Plasmodium falciparum | Immunofluorescence (V-NAR) | [55] |
| Trypanosomal pyruvate kinase | Trypanosoma congolense | ELISA and LFA | [35] |
| Paraflagellar rod protein | All trypanosome species (Trypanosoma evansi, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma vivax) | ELISA and immunofluorescence | [56] |
| Glycosomal aldolase | Trypanosoma congolense | ELISA | [57, 58] |
| Iron superoxide dismutase 1, tryparedoxin 1, nuclear transport factor 2 | Leishmania infantum | ELISA | [59] |
| Fasciola excretory secretory protein | Fasciola hepatica | ELISA | [60] |
| Taenia solium 14 kDa diagnostic glycoprotein | Taenia solium | Sandwich ELISA | [61] |
| Excretory secretory protein | Toxocara canis | Sandwich ELISA and electrochemical magnetosensor | [49, 50, 62] |
| Alternaria mycotoxin tenuazonic acid | Alternaria | CLEIA and BLEIA | [63] |
| Detection of bacterial infection | |||
| Chaperonin GroEL | Brucella | ELISA | [20] |
| Flagella | Campylobacter jejuni or Campylobacter coli | Fluorescence microscopy/immunoblotting | [64, 65] |
| Listeria monocytogenes | Sandwich ELISA | [66] | |
| Shiga toxin type 2 (B domain) | Shigella dysenteriae | Sandwich ELISA | [67] |
| Cholera toxin | Vibrio cholerae | V-NAR Sandwich ELISA | [68] |
| Detection of viral infection | |||
| Dengue virus type 2 NS1 protein | Dengue virus | LFA | [34] |
| H5N1 | Influenza H5N1 | Double nanobody Sandwich ELISA | [69] |
| E2/E3E2 envelope protein | Western equine encephalitis virus | Sandwich ELISA | [70] |
| FMDV 3ABC protein and synthetic peptides | Foot-and-mouth disease virus | Competitive ELISA | [71] |
| PEDV N protein | Porcine epidemic diarrhea virus | Blocking ELISA | [72] |
| Ebola virus nuclear protein | Zaire Ebola virus | V-NAR ELISA | [73] |
| Detection of small toxic molecules | |||
| Caffeine | Caffeine contamination | Competitive ELISA | [27] |
| Biphenyl 2,3-dioxygenase | Oil refinery waste treatment | Western blot | [36] |
| Parathion | Organophosphorous pesticide detection | One-step direct competitive fluorescent immunoassay | [37] |
| 3-Phenoxybenzoic acid | Detection of pyrethroid insecticides in urine | One-step direct competitive fluorescent immunoassay | [38] |
| Tetrabromobisphenol | Flame retardant | Competitive ELISA | [40] |
| Dicamba | Contamination with selective herbicide | CLEIA | [39] |
| Detection of human disease or malignancy | |||
| Human glycophorin A (CD235a) | Anti-HIV-1 p24 antibodies | Anti-CD235a VHH fused to HIV-1 p24–agglutination for HIV diagnosis | [74] |
| Alpha-fetoprotein | Cancer biomarker | ELISA and immuno-PCR | [75] |
| Pancreatic secretory zymogen-granule glycoprotein 2 | Crohn’s disease | ELISA and immunohistochemistry | [76] |
| CEA | Cancer biomarker | Biosensor for cancer biomarker | [44] |
| Procalcitonin | Serum marker for bacterial infections | Electrochemiluminescence | [43] |
| Growth hormone | Anti-doping assay | Sandwich ELISA | [48] |
| CD22 | B-cell malignancies/leukemia | ELISA and FACS | [77] |
| hPSA | Prostate cancer | Sandwich ELISA/Surface plasma resonance-based assay | [45, 46] |
| CD38 | Soluble CD38 in multiple myeloma | Sandwich assay | [78] |
| Human β-2-microglobulin | Amyloid disease | Fluorescence immunostaining | [79] |
| In vivo non-invasive imaging | |||
| EGFR | Tumor solid burden | SPECT/microCT | [80] |
| CD33 | Acute myeloid leukemia | Non-invasive imaging (PET/SPECT) | [81] |
| HER2 | Breast cancer | [54] | |
| MMR | Tumor-associated macrophages | SPECT/microCT and PET | [82, 83] |
| PSMA | Prostate cancer | SPECT/microCT | [84] |
| CD20 | Non-Hodgkin lymphoma | Theranostic | [85, 86] |
| DPP6 | Pancreatic endocrine cells | SPECT/CT | [87] |
| CA IX | Hypoxic ductal carcinoma | Molecular fluorescence imaging | [88] |
| Clec4F and Vsig4 | Kupffer cells, acute hepatitis, staging of liver pathogenesis | Immunohistochemistry and SPECT | [89] |
| CRIg | Rheumatoid arthritis/joint inflammation | SPECT/CT | [90, 91] |
| VCAM1 | Atherosclerotic lesions | SPECT imaging | [92] |
| Fibronectin | Breast cancer, melanoma | PET/CT | [93] |
Abbreviations: LFA, lateral flow assay; SPECT, single-photon emission computed tomography; CT, computed tomography; CLEIA, chemiluminescence enzyme immunoassay; BLEIA, bioluminescent enzyme immunoassay; MMR, macrophage mannose receptor; V-NAR, variable domain of immunoglobulin new antigen receptor. PEDV, porcine epidemic diarrhea virus; FMDV, foot-and-mouth disease virus; VHH, variable heavy chain-only antibodies (also known as single-domain antibodies); HIV, human immunodeficiency virus; PCR, polymerase chain reaction; FACS, fluorescent activated cell sorting; hPSA, human prostate specific antigen; EGFR, epidermal growth factor receptor; PET, positron emission tomography; CA IX, carbonic anhydrase 9; PSMA, prostate-specific membrane antigen; NS1, non-structural protein 1.