Rational design of antibodies and development of a novel method for (1-3)--D glucan detection as an alternative to Limulus amebocyte lysate assay

With advances in medicine, increasing medical interventions have increased the risk of invasive fungal disease development. (1-3)-beta-D glucan (BDG) is a common fungal biomarker in serological tests. However, the scarcity of Limulus resources for BDG detection poses a challenge. This study addresses the need for an alternative to Limulus amebocyte lysate by using BDG mutant antibody for chemiluminescence detection. The wild-type BDG antibody was obtained by immunizing rabbits. An optimal V52(H)I/N34(L) Y mutant antibody, which has increased 3.7-fold of the testing efficiency compared to the wild-type antibody, was first achieved by mutating "hot-spot" residues that contribute to strong non-covalent bonds, as determined by alanine scanning and molecular dynamics simulation. The mutant was then applied to develop the magnetic particle chemiluminescence method. 574 clinical samples were tested using the developed method, with a cutoff value of 95 pg/mL set by Limulus amebocyte lysate. The receiver operating characteristic curve demonstrated an area under the curve of 0.905 (95% CI: 0.880-0.929). Chemiluminescence detected an antigen concentration of 89.98 pg/mL, exhibiting a sensitivity of 83.33% and specificity of 89.76%. In conclusion, the results showed a good agreement with Limulus amebocyte lysate and demonstrated the feasibility of using BDG mutant antibodies for invasive fungal disease diagnosis. The new method based on chemiluminescence for detecting BDG could shorten the sample-to-result time to approximately 30 min, rescue Limulus from being endangered and is resource efficient in terms of equipment and the non-use of a skilled technician.