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An in-house ELISA method for measuring surfactant protein A

Cheong KB, Cheong SK, Boo NY, Jemilah M, Ton SH

Malays J Pathol, 1995 Dec;17(2):91-6.
PMID: 8935133

An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.

Matched MeSH terms: Proteolipids/blood*; Proteolipids/immunology; Proteolipids/standards
Sequence analysis and characterization of vacuolar-type H+ -ATPase proteolipid transcript from Acanthus ebracteatus Vah1

Ho CL, Nguyen PD, Harikrishna JA, Rahim RA

DNA Seq., 2008 Feb;19(1):73-7.
PMID: 17852357

The vacuolar-type H+ -ATPase (V-ATPase) is a multimeric enzyme with diverse functions in plants such as nutrient transport, flowering, stress tolerance, guard cell movement and development. A partial sequence of V-ATPase proteolipid was identified among the expressed sequence tags (ESTs) generated from Acanthus ebracteatus, and selected for full-length sequencing. The 876-nucleotide cDNA consists of an open reading frame of 165 amino acids. The deduced amino acid sequence displays high similarity (81%) with its homologs from Arabidopsis thaliana, Avecinnia marina and Gossypium hirsutum with the four transmembrane domains characteristics of the 16 kDa proteolipid subunit c of V-ATPase well conserved in this protein. Southern analysis revealed the existence of several members of proteolipid subunit c of V-ATPase in A. ebracteatus. The mRNA of this gene was detected in leaf, floral, stem and root tissues, however, the expression level was lower in stem and root tissues.

Matched MeSH terms: Proteolipids/genetics*; Proteolipids/chemistry
Surfactant protein A and stable microbubble formation in tracheal aspirates

Cheong KB, Cheong SK, Boo NY

Malays J Pathol, 1996 Dec;18(2):101-5.
PMID: 10879230

This study aimed to determine the role of surfactant protein A (SP-A) in the formation of stable microbubble in tracheal aspirates. Our results showed that as the concentration of anti SP-A antibodies added to tracheal aspirate specimens increased, the number of stable microbubble formed in the specimen decreased. The correlation between stable microbubble counts and the SP-A levels in the tracheal aspirates was good, r = 0.85, p < 0.05. This study suggests that SP-A plays an important role in stable microbubble formation. Measurement of small stable microbubbles is thus a useful bedside test for predicting the SP-A activity in the tracheal aspirates and in indirect measurement of lung maturity.

Matched MeSH terms: Proteolipids/immunology; Proteolipids/metabolism*
Isolation of human surfactant protein A from amniotic fluid

Cheong KB, Cheong SK, Boo NY, Jemilah M, Ton SH

Malays J Pathol, 1995 Dec;17(2):97-101.
PMID: 8935134

Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SP-A may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SP-A, we used a discontinuous sucrose density gradient to isolate SP-A from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SP-A from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SP-A were used as immunogens to raise antibodies in rabbits for the immunoassay.

Matched MeSH terms: Proteolipids/isolation & purification*
Usefulness of stable microbubble test of tracheal aspirate for the diagnosis of neonatal respiratory distress syndrome

Boo NY, Cheong KB, Cheong SK, Lye MS, Zulfiqar MA

J Paediatr Child Health, 1997 Aug;33(4):329-34.
PMID: 9323622

OBJECTIVES: To compare the overall accuracy of the stable microbubble test (SM test) with measurement of level of surfactant protein A (SP-A) of tracheal aspirate for the diagnosis of respiratory distress syndrome (RDS).
METHODOLOGY: Tracheal aspirates were obtained from neonates on ventilatory support. The SM test was carried out on specimens of tracheal aspirate immediately after collection. Levels of SP-A in tracheal aspirates were determined by enzyme-linked immunosorbent assay (ELISA) method. The results of the SM test and SP-A level of the tracheal aspirates were compared against the clinical diagnosis of RDS based on clinical, radiological and bacteriological findings.
RESULTS: Both the median microbubble counts (6 microbubbles/mm2, range = 0-90) and median SP-A levels (100 micrograms/L, range = 0-67447) of infants with RDS were significantly lower than those of infants with no obvious lung pathology (P < 0.0001), and pneumonia (P < 0.0001). The SM test of tracheal aspirates had higher overall accuracy for the diagnosis of RDS than measurement of SP-A levels (94.6% vs 82.4%). When the receiver operating characteristic (ROC) curves of both tests for RDS were compared, the area under the ROC curve of the SM test was larger (0.9689) than that of the SP-A method (0.8965).
CONCLUSIONS: This study showed that the SM test of tracheal aspirate was a useful bedside diagnostic test for RDS. It could be carried out at any time after birth on infants requiring ventilatory support.

Matched MeSH terms: Proteolipids/analysis*
Mycobacterium smegmatis proteoliposome induce protection in a murine progressive pulmonary tuberculosis model

Tirado Y, Puig A, Alvarez N, Borrero R, Aguilar A, Camacho F, et al.

Tuberculosis (Edinb), 2016 12;101:44-48.
PMID: 27865396 DOI: 10.1016/j.tube.2016.07.017

Tuberculosis (TB) remains an important cause of mortality and morbidity. The TB vaccine, BCG, is not fully protective against the adult form of the disease and is unable to prevent its transmission although it is still useful against severe childhood TB. Hence, the search for new vaccines is of great interest. In a previous study, we have shown that proteoliposomes obtained from Mycobacterium smegmatis (PLMs) induced cross reactive humoral and cellular response against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLMs, a murine model of progressive pulmonary TB was used. Animals immunized with PLMs with and without alum (PLMs/PLMsAL respectively) showed protection compared to non-immunized animals. Mice immunized with PLMsAL induced similar protection as that of BCG. Animals immunized with BCG, PLMs and PLMsAL showed a significant decrease in tissue damage (percentage of pneumonic area/lung) compared to non-immunized animals, with a more prominent effect in BCG vaccinated mice. The protective effect of the administration of PLMs in mice supports its future evaluation as experimental vaccine candidate against Mtb.

Matched MeSH terms: Proteolipids/immunology*