Development of a Double-Antibody Sandwich ELISA for Rapid Detection of the MCP Antigen Concentration in Inactivated ISKNV Vaccines

Liang, Hongru; Li-xi, Zhang; Fu, Xiaozhe; Lin, Qiang; Liu, Lihui; Niu, Yinjie; Luo, Xian; Huang, Zhibin; Li, Ningqiu

doi:10.3390/vaccines9111264

“…The optimal curves and related formulas y = 1.696-0.087x+3.100/x 2 and y = -0.205+0.587x-0.097x 2 +0.001x 3 drawn by Curve Expert software also fit the classical curve equations of ELISA competition method and double antibody sandwich method well. 14,15 The determination coefficients r 2 of both curves were greater than 0.99, providing quality assurance for the overall linear range of LAM ELISA in different populations in the later stage.…”

Section: Discussionmentioning

confidence: 84%

Analysis of the Validity of Urine LAM ELISA for Tuberculosis Infection

Junpeng,

Avoi,

Atil

2024

IJPQA

0

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Objective: To explore the validity of urinary lipoarabinomannan (LAM) enzyme-linked immunosorbent assay (ELISA) assay technology for detecting MTB infection in the double infection of acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus-tuberculosis (HIV-TB) population with sputum-producing problems, and to explore the background value and medical reference value range of urinary LAM in the general population and people living with human immunodeficiency virus (HIV) or patients with acquired immunodeficiency syndrome (HIV/AIDS population). Method: About 307 individuals from the general population group, HIV/AIDS population group, TB population group and HIV-TB population group provided by Seventh Hospital of Tangshan city were selected for early morning urine analysis. LAM ELISA competition method and double antibody sandwich method were used to detect the concentration of LAM in urine. Standard curves of LAM optical density OD value were drawn. The differences in LAM concentration in different groups of urine were calculated, and the diagnostic validity of LAM ELISA techniques was explored. Result: (1) The corresponding curve formula of the ELISA competition method was y = 1.696-0.087x+3.100/x2; The corresponding curve formula for the double antibody sandwich method was y = -0.205+0.587x-0.097x2+0.001x3. (2) In LAM ELISA competition method, the difference in LAM OD values between the TB population group and the general population group was statistically significant (t = 3.393, p &LT; 0.05), and the difference in LAM OD values between the HIV-TB population group and the HIV/AIDS population group was statistically significant (t = 2.294, p &LT; 0.05); The difference in LAM concentration between TB population group and general population group was statistically significant (t = -4.642, p &LT; 0.05), and the difference in LAM concentration between HIV-TB population group and HIV/AIDS population group was statistically significant (t = -4.737, p &LT; 0.05). In LAM ELISA double antibody sandwich method, there was a statistically significant difference in LAM OD values between TB population group and the general population group (t = -2.566, p &LT; 0.05), and there was a statistically significant difference in LAM OD values between HIV-TB population group and HIV/AIDS population group (t = -3.212, p &LT; 0.05); The difference in LAM concentration between TB population group and general population group was statistically significant (t = -5.722, p &LT; 0.05), and the difference in LAM concentration between HIV-TB population group and HIV/AIDS group was statistically significant (t = -8.118, p &LT; 0.05). (3) Receiver operating characteristic curve (ROC) curve analysis showed that in the LAM ELISA competition method, the SPE of TB infection in the HIV-TB population group diagnosed with urine LAM was higher than those in TB population group, with a statistically significant difference (F = 31.227, p &LT; 0.05). Compared to the general population group, LAM ELISA competition method SEN in TB population group was lower than that in the ELISA double antibody sandwich method, and the difference was statistically significant (F = 15.667, p &LT; 0.05). Conclusion: The validity of urine LAM ELISA technology in the HIV-TB population group with TB infection was better than that in TB population group, and the validity of the LAM ELISA double antibody sandwich method was better than that in ELISA competitive method. The feasibility of urine LAM ELISA technology in HIV-TB was worthy of recognition, and the technology could be further improved and promoted.

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“…In developing double antibody S-ELISA for the detection of specific antigen, mAb-based assays are quite stable and specific because they are monospecific against the unique epitopes of the test antigen [69]. For a food-borne pathogen like Brucella, the medium of dairy products is highly complex and the availability of the bacteria is also too low to detect.…”

Section: Discussionmentioning

confidence: 99%

Immunoassay-based evaluation of rOmp28 protein as a candidate for the identification of Brucella species

Hans

¹

,

Thavaselvam

²

2023

Journal of Medical Microbiology

1

0

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Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus , cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden. Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella . Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection. Methodology. We purified recombinant rOmp28 with Ni–NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella . Checkerboard sandwich ELISA and P/N ratio (optical density of ‘P’ positive test sample to ‘N’ negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella . Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml−1) and rOmp28-derived mice IgG (detection antibody at 100 µg ml−1) with a detection range of 102 to 108 cells ml−1 and a limit of detection at 102 cells ml−1. A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria. Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.

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