A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivaxdeveloped from two lactate dehydrogenase protein segments

Abstract

BackgroundImmunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.MethodsTwo recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.Results“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.ConclusionBoth tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.

Simultaneous Quantification of Plasmodium Antigens and Host Factor C-Reactive Protein in Asymptomatic Individuals with Confirmed Malaria by Use of a Novel Multiplex Immunoassay

“…A new diagnostic tool that can quantify the two most commonly used antigens in malaria RDTs, HRP2 and pLDH, as well as the inflammation marker CRP, from the same blood sample was developed. While assays for pLDH or highly sensitive assays for HRP2 have been described previously (16, 26, 27), the 4-plex array is capable of measuring both HRP2 and pLDH antigens at low concentrations and discriminating between two Plasmodium species with no cross-reactivity (Table 2). The 4-plex array is comparable in LOD to these highly sensitive bead-based assays for HRP2, which range in LOD from 0.24 pg/ml to 70 pg/ml, depending on the HRP2 type detected (16).…”

Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei

“…Sandwich ELISA was carried out as per the protocol described elsewhere [37] with modifications. ELISA plates were coated with 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1:100 dilution of rabbit anti-flagellin antibodies overnight at 4 C. After washing three times with PBST the wells were blocked with 3% skimmed milk powder in PBS at 37 C for 2 h. Cells from 10 0 e10 9 cfu/ml were added in duplicates to the wells of the ELISA plate and were incubated for 2 h at 37 C. The wells, washed thoroughly and 1:200 dilution of chicken anti-flagellin antibodies, were incubated at 37 C for 2 h. The rabbit anti-chicken HRPO conjugate was added at 1:5000 dilution and incubated at 37 C for 1 h. After thorough washing, the plate was developed using OPD-H 2 O 2 and incubated at room temperature for 10 min and the reading was recorded at 492 nm in an ELISA reader.…”

An Anti-proteome Nanobody Library Approach Yields a Specific Immunoassay for Trypanosoma congolense Diagnosis Targeting Glycosomal Aldolase

“…Where nucleic acid-based assays are unaffordable and sensitivity of microscopy becomes limited, enzyme-linked immunosorbent assay (ELISA) can be used as an alternative tool for detection of infectious diseases through antibody [ 8 ] or antigen [ 9 ] detection. ELISA is a robust technique which operates without sophisticated equipments therefore favoring its application in less well-established laboratories such as those found in developing countries.…”