ImmuLisa Enhanced Gliadin IgA Antibody ELISA, ImmuLisa Enhanced Gliadin IgG Antibody ELISA

K163177 · Immco Diagnostics, Inc. · MST · Jul 28, 2017 · Immunology

Device Facts

• Record ID: K163177
• Device Name: ImmuLisa Enhanced Gliadin IgA Antibody ELISA, ImmuLisa Enhanced Gliadin IgG Antibody ELISA
• Applicant: Immco Diagnostics, Inc.
• Product Code: MST · Immunology
• Decision Date: Jul 28, 2017
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5750
• Device Class: Class 2

Intended Use

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA: Enzyme linked immunosorbent assays (ELISA) for the qualitative or semi-quantitative detection of IgA anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease or dermatitis herpetiformis in conjunction with other laboratory and clinical findings. ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA: Enzyme linked immunosorbent assays (ELISA) for the qualitative or semi-quantitative detection of IgG anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease or dermatitis herpetiformis in conjunction with other laboratory and clinical findings.

Device Story

Solid-phase ELISA for qualitative detection of IgA or IgG anti-gliadin antibodies in human serum; aids diagnosis of celiac disease or dermatitis herpetiformis. Microwells coated with native purified gliadin antigen; patient serum, calibrators, and controls incubated; specific antibodies bind to antigen. Bound antibodies detected via enzyme-labeled anti-human IgA or IgG conjugate and TMB substrate; color change measured by spectrophotometer at 450 nm. Results expressed in EU/ml; reported as positive or negative. Used in clinical laboratories by trained personnel. Output assists clinicians in identifying celiac disease, particularly in IgA-deficient patients where other markers may be unreliable.

Clinical Evidence

Clinical validation included 751 serum samples (295 target disease group: celiac disease/dermatitis herpetiformis; 456 non-target disease group). Sensitivity for celiac disease ranged from 55.6% to 76.4% depending on antibody type and indeterminate result classification. Specificity ranged from 95.4% to 98.5%. Method comparison against predicate showed total percent agreement of 92.0%–95.6%.

Technological Characteristics

Solid-phase immunoassay using polystyrene microplate wells. Energy source: spectrophotometer (450 nm). Materials: purified native gliadin antigen, HRP-conjugated anti-human IgA/IgG, TMB substrate, sulfuric acid stop solution. Manual assay format. Storage: 2-8°C. Shelf-life: 18 months. No connectivity; standalone laboratory test.

Indications for Use

Indicated for the qualitative or semi-quantitative detection of IgA or IgG anti-gliadin antibodies in human serum to aid in the diagnosis of celiac disease or dermatitis herpetiformis in conjunction with other clinical and laboratory findings.

Regulatory Classification

Identification

A radioallergosorbent immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the allergen antibodies (antibodies which cause an allergic reaction) specific for a given allergen. Measurement of specific allergen antibodies may aid in the diagnosis of asthma, allergies, and other pulmonary disorders.

Special Controls

*Classification.* Class II (special controls). The device, when intended to detect any of the allergens included in Table 1 in this paragraph, is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 866.9. Table 1—Class II Exempt Allergens Under § 866.5750—Radioallergosorbent (RAST) Immunological Test Systems Allergen code Allergen product Source (taxonomical name) Grass Pollens g1 Sweet vernal grass Anthoxanthum odoratum. g3 Cocksfoot grass, Orchard grass Dactylis glomerata. g4 Meadow fescue Festuca elatior. g5 Rye-grass (perennial rye grass) Lolium perenne. g7 Common reed (common reed grass) Phragmites communis. g8 Meadow grass, Kentucky blue (June grass) Poa pratensis. g9 Redtop, Bentgrass Agrostis stolonifera ,Agrostis gigantea (Agrostis alba ). g11 Brome grass Bromus inermis. g12 Cultivated rye (cultivated rye grass) Secale cereale. g13 Velvet grass Holcus lanatus. g14 Cultivated oat (cultivated oat grass) Avena sativa. g15 Cultivated wheat (cultivated wheat grass) Triticum aestivum (Triticum spp.). g16 Meadow foxtail (meadow foxtail grass) Alopecurus pratensis. g17 Bahia grass Paspalum notatum. g24 Wheat grass, Western Agropyron smithii (Elymus smithii ). g30 Bluegrass, annual Poa annua. g70 Wild rye grass Elymus triticoides Elymus condensatus. g71 Canary grass Phalaris arundinacea. g201 Barley, cultivated Hordeum vulgare. g202 Maize, corn (cultivated corn) Zea mays. g203 Salt grass Distichlis spicata. g204 False oat-grass Arrhenatherum elatius. g216 Cyn d 1 Cynodon dactylon. g701 Phl p 1.0102, Phl p 5.0101 Phleum pratense. g702 Phl p 7.0101 Phleum pratense. g703 Phl p 12.0101 Phleum pratense. Weed Pollens w2 Western ragweed Ambrosia psilostachya. w4 False ragweed Ambrosia acanthicarpa (Franseria acanthicarpa ). w5 Wormwood Artemisia absinthium Artemisia annua. w6 Mugwort Artemisia vulgaris. w7 Marguerite, ox-eye daisy Chrysanthemum leucanthemum. w8 Dandelion Taraxacum vulgare ,Taraxacum officinale. w9 Plantain (English), Ribwort Plantago lanceolata. w10 Goosefoot, lamb's quarters Chenopodium album. w11 Saltwort (prickly), Russian thistle Salsola kali (Salsola pestifer ). w12 Goldenrod Solidago virgaurea (Solidago spp.). w13 Cocklebur, common Xanthium commune. w14 Common pigweed (rough pigweed) Amaranthus retroflexus. w15 Scale, Lenscale Atriplex lentiformis. w16 Rough marsh elder Iva ciliate ,Iva annua. w17 Firebush (Kochia) Kochia scoparia. w18 Sheep sorrel Rumex acetosella. w19 Wall pellitory Parietaria officinalis. w20 Nettle (Common stinging nettle) Urtica dioica. w21 Wall pellitory Parietaria judaica. w22 Japanese hop (careless weed) Humulus japonicas (Humulus scandens ). w23 Yellow dock, Yellow dockweed Rumex crispus. w24 Spiny pigweed Amaranthus spinosus. w27 Carnation Dianthus spp. w28 Rose Rosa rugosa. w33 Clover Trifolium pratense. w35 Mexican tea Chenopodium ambrosioides. w36 Rabbit bush Ambrosia deltoidea (Franseria deltoides ). w37 Salt bush, annual Atriplex wrightii. w39 Water hemp, Western Amaranthus rudis (Acnida tamariscina ). w41 Burrobrush Hymenoclea salsola. w42 Poverty weed Baccharis neglecta. w43 Common sagebrush Artemisia tridentata. w45 Alfalfa Medicago sativa. w46 Dog fennel Eupatorium capillifolium. w53 Geranium Geranium spp. w67 Groundsel bush Baccharis halimifolia. w69 Iodine bush Allenrolfea occidentalis. w70 Ragweed, slender Ambrosia confertiflora. w75 Wing scale (wingscale) Atriplex canescens. w82 Careless weed Amaranthus palmeri ,Amaranthus hybridus. w90 Japanese hop Humulus japonicas (Humulus scandens ). w203 Rape (rape pollen) Brassica napus. w204 Sunflower Helianthus annuus. w206 Camomile Matricaria chamomilla. w207 Lupin Lupinus spp. w210 Sugar-beet Beta vulgaris. w211 Par j 2.0101 Parietaria judaica. w231 Art v 1 Artemisia vulgaris (Mugwort). w232 Sal k 1 Salsola kali. w233 Art v 3 Artemisa vulgaris (LTP, Mugwort). w234 Pla l 1 Plantago lanceolata. w235 Che a 1.0101 Chenopodium album. w236 Mer a 1.0101 Mercurialis annua. a753 Art v 1 Artemisia vulgaris (Mugwort weed). Tree Pollens t1 Box-elder (Maple) Acer negundo ,Acer saccharum. t2 Gray alder, speckled alder (alder) Alnus incana. t4 Hazel, hazelnut Corylus avellana ,Corylus americana. t5 American beech (beech) Fagus grandifolia (Fagus americana ). t6 Mountain juniper, Mountain cedar Juniperus ashei (Juniperus sabinoides ). t8 Elm Ulmus americana. t9 Olive Olea europaea. t10 Walnut Juglans californica ,Juglans nigra. t11 Maple leaf sycamore, London plane, Plane tree Platanus acerifolia. t61 Sycamore Platanus occidentalis. t12 Willow Salix caprea ,Salix nigra. t14 Cottonwood (Eastern Cottonwood/Black Cottonwood) Populus deltoides. t15 White ash Fraxinus americana. t16 White pine Pinus strobus. t18 Eucalyptus, gum-tree Eucalyptus globulus (Eucalyptus spp.). t19/t26 Acacia Acacia longifolia (Acacia spp.). t20 Mesquite Prosopis glandulosa/ Prosopis juliflora. t21 Melaleuca, cajeput tree Melaleuca quinquenervia (Melaleuca leucadendron ). t22 Pecan, hickory Carya illinoinensis (Carya pecan ). t23 Italian/Mediterranean/funeral cypress Cupressus sempervirens. t24 Japanese cypress Chamaecyparis obtusa (Chamaecyparis spp.). t25 Ash Fraxinus excelsior. t27 Maple, red Acer rubrum. t29 Acacia Acacia spp. t30 Birch, white Betula populifolia. t32 Willow, black Salix nigra. t33 Ash, Arizona Fraxinus velutina. t35 Cedar, salt Tamarix gallica. t37 Bald cypress (white bald cypress) Taxodium distichum. t38 Elm, Chinese/Siberian Ulmus pumila. t40 Hazelnut tree Corylus americana. t41 White hickory Carya alba (Carya tomentosa ). t42 Oak, red Quercus rubra. t43 Loblolly pine Pinus taeda. t44 Hackberry Celtis occidentalis. t45 Cedar elm Ulmus crassifolia. t47 Juniper, one seed Juniperus monosperma. t48 Pine, lodgepole Pinus contorta. t49 Pine, ponderosa Pinus ponderosa. t50 Beech, European Fagus sylvatica. t51 Tree of Heaven Ailanthus altissima. t52 Western white pine Pinus monticola. t54 Russian olive Elaeagnus angustifolia. t55 Scotch broom Cytisus scoparius. t56 Bayberry Myrica cerifera. t57 Red cedar Juniperus virginiana. t60 Western juniper Juniperus occidentalis. t61 Sycamore Platanus occidentalis. t70 Mulberry (white mulberry) Morus alba. t71 Red mulberry Morus rubra. t72 Queen palm Arecastrum romanzoffiamon. t73 Australian pine Casuarina equisetifolia. t77 Oak mix (red, white, black) Quercus spp. t80 Japanese cypress Chamaecyparis obtusa. t81 Japanese alder Alnus japonica. t83 Mango tree Mangifera indica. t90 Walnut, black Juglans nigra. t96 Poplar, white (poplar) Populus alba. t103/t218 Virginia live oak (live oak) Quercus virginiana. t105 Pepper tree Schinus molle. t110 Orange tree Citrus sinensis. t201 Spruce, Norway spruce Picea abies (Picea excelsa ). t202 Alder, smooth Alnus incana spp.Rugosa (Alnus rugosa ). t203 Horse chestnut Aesculus hippocastanum. t205 Elder Sambucus nigra. t206 Chestnut Castanea sativa. t207 Douglas fir Pseudotsuga menziesii (Pseudotsuga taxifolia ). t208 Linden Tilia cordata. t209 Horn beam Carpinus betulus. t210 Privet Ligustrum vulgare. t211 Sweet gum Liquidambar styraciflua. t212 Cedar Libocedrus decurrens. t213 Pine Pinus radiata. t214 Date palm Phoenix canariensis. t215 Lilac Syringa vulgaris. t217 Pepper tree Schinus molle. t217 Red alder Alnus rubra. t218 Virginia live oak Quercus virginiana. t218 Bayberry (bayberry/sweet gale) Myrica gale. t219 Palo verde Cercidium floridum. t219 Red cedar Juniperus virginiana. t220 Bet v 4 Betula verrucosa (Birch). t221 Bet v 2.0101, Bet v 4 Betula verrucosa (Birch). t222 Cypress (Arizona cypress) Cupressus arizonica. t223 Oil palm Elaeis guineensis. t224 Ole e 1 Olea europaea. t225 Bet v 6 Betula verrucosa (Birch). t226 Cup a 1 Cupressus arizonica. t227 Ole e 7 Olea Europaea. t228 Aspen, quaking Populus tremuloides. t229 Eastern hemlock Tsuga canadensis. t230 Redwood (sequoia) Sequoia sempervirens. t232 Pussy willow Salix discolor. t240 Ole e 9.0101 Olea Europaea. t241 Pla a 1.0101 Platanus acerifolia. t242 Pla a 2 Platanus acerifolia. t243 Pla a 3.0101 Platanus acerifolia. t244 Cor a 1.0103 Corylus avellana. t245 Aln g 1.0101 Alnus glutinosa. t246 Cry j 1 Cryptomeria japonica. t280 Locust tree Robinia pseudoacacia. t401 Brazilian peppertree Schinus terebinthifolius. t402 Mastic tree Pistacia lentiscus. t404 Tree of heaven Ailanthus altissima. t406 Date palm Phoenix dactylifera. a482 Ole e 1 Olea europaea (Olive Oil). Mites d207 Blo t 5.0101 Blomia tropicalis. d208 Lep d 2.0101 Lepidoglyphus destructor. Microorganisms, Molds, Yeast m1 Penicillium chrysogenum (Penicillium notatum ) Penicillium chrysogenum (Penicillium notatum ). m2 Cladosporium herbarum (Hormodendrum ) Cladosporium herbarum (Hormodendrum ). m3 Aspergillus fumigatus Aspergillus fumigatus. m4 Mucor racemosus Mucor racemosus. m5 Candida albicans Candida albicans. m7 Botrytis cinerea Botrytis cinerea. m8 Drechslera halodes (Setomelanomma rostrata, Helminthosporium halodes, Helminthosporium interseminatum ) Drechslera halodes (Setomelanomma rostrata, Helminthosporium halodes. m9 Fusarium moniliforme (Fusarium proliferatum ) Fusarium moniliforme (Fusarium proliferatum ). m10 Stemphylium botryosum Stemphylium herbarum (Stemphylium botryosum ). m11 Rhizopus nigricans Rhizopus nigricans. m12 Aureobasidium pullulans Aureobasidium pullulans. m13 Phoma betae Phoma betae. m14 Epicoccum purpurascens Epicoccum purpurascens (Epicoccum nigrum ). m15 Trichoderma viride Trichoderma viride. m16 Curvularia lunata Curvularia lunata ,Curvularia specifera (K923044 ). m17 Cladosporium fulvum Cladosporium fulvum. m18 Fusarium culmorum Fusarium culmorum. m19 Aspergillus versicolor Aspergillus versicolor. m20 Mucor mucedo Mucor mucedo. m21 Aspergillus clavatus Aspergillus clavatus. m22 Micropolyspora faeni Saccharopolyspora rectivirgula (Micropolyspora faeni ). m23 Thermoactinomyces vulgaris Thermoactinomyces vulgaris. m24 Stachybotrys atra Stachybotrys chartarum (Stachybotrys atra ). m24 Paecilomyces spp Paecilomyces spp. m25 Aspergillus versicolor Aspergillus versicolor. m25 Penicillium brevicompactum Penicillium brevicompactum. m26 Cladosporium cladosporioides Cladosporium cladosporioides. m26 Penicillium citrinum Penicillium citrinum. m27 Penicillium spp Penicillium spp. m29 Aspergillus repens Aspergillus repens. m30 Penicillium roqueforti Penicillium roqueforti. m32 Cladosporium cladosporioides Cladosporium cladosporioides. m34 Serpula lacrymans Serpula lacrymans. m36 Aspergillus terreus Aspergillus terreus. m37 Trichophyton mentagrophytes Trichophyton mentagrophytes. m40 Aspergillus amstelodami Aspergillus amstelodami. m43 Saccharomyces Carlsberg Saccharomyces carlsbergensis. m44 Saccharomyces cerevisiae Saccharomyces cerevisiae. m45 Hormodendrum hordei Hormodendrum hordei. m46 Bipolaris spicifera Bipolaris spicifera. m47 Aspergillus nidulans Aspergillus nidulans. m48 Aspergillus oryzae Aspergillus oryzae. m49 Fusarium oxysporum Fusarium oxysporum. m50 Micropolyspora faeni Saccharopolyspora rectivirgula (Micropolyspora faeni ). m51 Thermoactinomyces vulgaris Thermoactinomyces vulgaris. m53 Microspora canis Microsporum canis (Microspora canis ). m54 Aspergillus flavus Aspergillus flavus. m63 Helminthosporium intersemin Helminthosporium intersemin. m66 Mucor plumbeus Mucor plumbeus. m67 Mycogone Mycogone perniciosa. m68 Nigrospora oryzae Nigrospora oryzae. m69 Rhodotorula Rhodotorula rubra (Rhodotorula mucilaginosa ). m70 Malassezia furfur (Pityrosporum orbiculare ) Malassezia furfur (Pityrosporum orbiculare ). m71 Spondylocladium Spondylocladium spp. m72 Epidermophyton Epidermophyton floccosum. m73 Epicoccum nigrum Epicoccum purpurascens (Epicoccum nigrum ). m80 Staphylococcal enterotoxin A (Sta a SEA ) Staphylococcus aureus. m80 Helminthosporium spp Helminthosporium spp. m81 Staphylococcal enterotoxin B (Sta a SEB ) Staphylococcus aureus. m88 Stemphylium solani Stemphylium solani. m93 Gliocladium fimbriatum Gliocladium fimbriatum. m94 Phycomyces blakesleeanus Phycomyces blakesleeanus. m201 Tilletia tritici (Ustilago nuda, Ustilago tritici ) (Barley smut) Tilletia tritici (Ustilago nuda, Ustilago tritici ). m202 Acremonium kiliense (Cephalosporium acremonium ) Acremonium kiliense (Cephalosporium acremonium ). m203 Trichosporon pullulans Trichosporon pullulans. m204 Ulocladium chartarum Ulocladium chartarum. m205 Trichophyton rubrum Trichophyton rubrum. m207 Aspergillus niger Aspergillus niger. m208 Chaetomium globosum Chaetomium globosum. m209 Penicillium frequentans Penicillium glabrum (Penicillium frequentans ). m209 Stachybotrys chartarum Stachybotrys chartarum (Stachybotrys atra ). m210 Trichophyton mentagrophytes var. goetzii Trichophyton mentagrophytes var. goetzii. m211 Trichophyton mentagrophytes var. interdigitale Trichophyton mentagrophytes var. interdigitale. m211 Oat smut Ustilago avenae. m212 Micropolyspora faeni Saccharopolyspora rectivirgula (Micropolyspora faeni ). m212 Geotrichum candidum Geotrichum candidum. m213 Bermuda grass smut Ustilago cynodontis. m214 Johnson grass smut Sphacelotheca cruenta. m215 Corn smut Ustilago maydis. m218 Asp f 1.0101 Aspergillus fumigatus. a3050 Asp r 1 Aspergillus restrictus. m219 Asp f 2 Aspergillus fumigatus. m220 Asp f 3.0101 Aspergillus fumigatus. m221 Asp f 4 Aspergillus fumigatus. m222 Asp f 6.0101 Aspergillus fumigatus. m223 Staphylococcal enterotoxin C (Sta a SEC ) Staphylococcus aureus. m224 Staphylococcal enterotoxin D (Sta a SED ) Staphylococcus aureus. m226 Staphylococcal enterotoxin TSST (Sta a TSST ) Staphylococcus aureus. m227 Malassezia spp. (Pityrosporum spp.) Malassezia spp. (Pityrosporum spp.). m228 Aspergillus flavus. m229 Alt a 1.0101 Alternaria alternata (Alternaria tenuis ). m230 Alt a 6.0101 Alternaria alternata (Alternaria tenuis ). m231 Cla h 8.0101 Cladosporium herbarum (Hormodendrum ). m300 Eurotium spp Eurotium spp. m304 Aspergillus oryzae Aspergillus oryzae. m305 Penicillium brevicompactum Penicillium brevicompactum. m309 Aspergillus terreus Aspergillus terreus. m310 Aspergillus nidulans Aspergillus nidulans. m311 Aspergillus flavus Aspergillus flavus. m312 Aspergillus clavatus Aspergillus clavatus. Epidermal & Animal e6 Guinea pig epithelium Cavia porcellus. e7 Pigeon droppings Columba palumbus ,Columba livia. e25 Chicken serum Gallus domesticus (Gallus gallus domesticus; Gallus spp.). e26 Parrot serum Psittacoidea spp. e62 Camel Camelus dromedaries. e70 Goose feathers Anser anser. e71 Mouse epithelium Mus musculus (Mus spp.). e73 Rat epithelium Rattus norvegicus. e74 Rat urine proteins Rattus norvegicus ,Rattus rattus. e75 Rat serum proteins Rattus norvegicus ,Rattus rattus. e76 Mouse serum proteins Mus musculus (Mus spp.). e77 Budgerigar droppings Melopsittacus undulatus. e78 Budgerigar feathers Melopsittacus undulatus. e79 Budgerigar serum proteins Melopsittacus undulatus. e80 Goat epithelium Capra hircus. e81 Sheep epithelium Ovis aries (Ovis spp.). e82 Rabbit epithelium Oryctolagus cuniculus (Oryctolagus spp.). e83 Swine epithelium Sus scrofa (Sus scrofa domesticus; Sus spp.). e84 Hamster epithelium Cricetus cricetus, Mesocricetus auratus, and Phodopus sungorus. e85 Chicken feathers Gallus domesticus (Gallus gallus domesticus; Gallus spp.). e86 Duck feathers Anas platyrhynchos. e87 Rat epithelium, serum proteins, and urine proteins Rattus norvegicus Rattus rattus. e88 Mouse epithelium, serum proteins, and urine proteins (mouse) Mus musculus (Mus spp.). e89 Turkey feathers Meleagris gallopavo. e90 Budgerigar serum proteins, feathers, and droppings Melopsittacus undulatus. e91 Pigeon serum proteins, feathers, and droppings Streptopelia roseogrisea ,Psittacidae spp. e92 Parrot serum proteins, feathers, and droppings Ara spp. e93 Pigeon serum proteins Streptopelia roseogrisea. e94 Fel d 1.0101 Felis domesticus. a345 Fel d 1 Felis domesticus. e98 Parrot droppings Psittacoidea spp. e101 Can f 1.0101 Canis familiaris (Canis domesticus ). a174 Can f 1 Canis familiaris (Canis domesticus ). e102 Can f 2.0101 Canis familiaris (Canis domesticus ). e196 Parakeet feathers Nymphicus hollandicus. e197 Parakeet droppings Nymphicus hollandicus. e198 Parakeet serum Nymphicus hollandicus. e199 Canary bird serum Serinus canarius. e200 Canary bird droppings Serinus canarius. e201 Canary bird feathers (Canary feathers) Serinus canarius. e202 Reindeer epithelium Rangifer tarandus. e203 Mink epithelium Mustela spp. e204 Bos d 6 Bos domesticus (Bos taurus; Bos spp.). e205 Horse, serum proteins Equus caballus (Equus spp.). e206 Rabbit, serum proteins Oryctolagus cuniculus (Oryctolagus spp.). e208 Chinchilla epithelium Chinchilla laniger. e209 Gerbil epithelium Meriones unguiculatus. e210 Fox epithelium Vulpes vulpes. e211 Rabbit, urine proteins Oryctolagus cuniculus (Oryctolagus spp.). e212 Swine, urine proteins Sus scrofa (Sus scrofa domesticus; Sus spp.). e213 Parrot feathers Ara spp. e214 Finch feathers Lonchura domestica. e215 Pigeon feathers Streptopelia roseogrisea (Streptopelia spp.),Columbia spp. e216 Deer epithelium Dama dama. e217 Ferret epithelium Mustela putorius. e218 Chicken droppings Gallus domesticus (Gallus gallus domesticus; Gallus spp.). e219 Chicken, serum proteins Gallus domesticus (Gallus gallus domesticus; Gallus spp.). e220 Fel d 2, Cat serum albumin Felis domesticus. e221 Can f 3 Canis familiaris (Canis domesticus ) (Dog serum albumin ). e222 Swine serum albumin (Sus s PSA) Sus scrofa (Sus scrofa domesticus; Sus spp.). e225 Lovebird feathers Psittacoidea agapomis. e226 Can f 5.0101 Canis familiaris. e227 Equ c 1.0101 Equus caballus. e228 Fel d 4.0101 Felis domesticus. e230 Equ c 3 Equus caballus. e231 Mus m 1 Mus musculus. Food f9 Rice Oryza sativa. f12 Pea (green pea) Pisum sativum. f15 White bean Phaseolus vulgaris. f19 Cayenne pepper Capsicum frutescens (Capsicum annum ). f21 Sugar cane Saccharum officinarum. f22 Raspberry Rubus idaeus. f26 Pork Sus scrofa (Sus scrofa domesticus; Sus spp.). f29 Watermelon Citrullus lanatus (Citrullus vulgaris ). f31 Carrot Daucus carota. f32 Oyster mushroom Pleurotus ostreatus. f33 Orange Citrus sinensis. f35 Potato Solanum tuberosum. f43 Mother's milk Homo sapiens. f44 Strawberry Fragaria vesca (Fragaria spp.). f45 Yeast, baker's Saccharomyces cerevisiae. f46 Pepper, Red Capsicum annuum. f47 Garlic Allium sativum. f48 Onion Allium cepa. f49 Apple Malus x domestica (Malus spp.). f51 Bamboo shoot Phyllostachys pubescens. f52 Cacao/chocolate Theobroma cacao. f54 Sweet potato Ipomoea batatas. f55 Common millet Panicum miliaceum. f56 Foxtail millet Setaria italica. f57 Japanese millet Echinochloa crus-galli. f58 Pacific squid Todarodes pacificus. f59 Octopus Octopus vulgaris (Octopus spp.). f63 Kefir NA. f67 Parmesan cheese NA. f81 Cheese, cheddar type NA. f82 Cheese, mold type NA. f83 Chicken Gallus domesticus (Gallus gallus domesticus; Gallus spp.). f86 Parsley Petroselinum crispum. f87 Melon Cucumis melo Cucumis melo + Citrullus lanatus. f88 Mutton (lamb) Ovis aries (Ovis spp.). f90 Malt Hordeum vulgare. f92 Banana Musa spp. f93 Cacao Theobroma cacao. f94 Pear Pyrus communis (Pyrus spp.). f97 Yam Dioscorea spp.Dioscorea opposita. f97 Chamomile tea Matricaria chamomilla. f98 Gliadin Triticum aestivum (Triticum spp.). f102 Cantaloupe Cucumis melo var. cantalupensis. f105 Chocolate Theobroma cacao. f109 Cottonseed Gossypium hirsutum. f110 Giant radish Raphanus sativus. f118 Zucchini Cucurbita pepo. f119 Radish Raphanus sativus. f120 Venison Capreolus capeolus. f121 Pinto bean Phaseolus vulgaris. f122 Cheese, American NA. f127 Black-eyed pea Vigna unguiculata. f131 Black Olive Olea europaea. f136 Red beet Beta vulgaris var. conditiva. f139 Goat's Cheese Capra aegagrus. f140 Bran NA. f141 Corn (vegetables) Zea mays. f152 Green bell pepper Capsicum annuum. f155 Brewer's yeast Saccharomyces carlsbergensis. f157 Duck Anas domesticus. f158 Goose Anser anser. f160 Camembert cheese NA. f162 Nectarine Prunus persica var. nucipersica. f163 Kohlrabi Brassica oleracea var. gongylodes. f65 Perch f166 Leek Allium porrum. f170 Cheese (Switzerland) (Swiss cheese) NA. f174 Fig Ficus carica. f177 Cranberry Vaccinium macrocarpon. f179 Raisin Vitis spp. f182 Lima bean Phaseolus lunatus. f198 Flaxseed (bruised grain) Linum usitatissimum. f199 Untreated native milk Bos domesticus (Bos taurus; Bos spp.). f208 Lemon Citrus limon. f209 Grapefruit Citrus paradisi. f210 Pineapple Ananas comosus. f211 Blackberry Rubus fruticosus. f212 Mushroom (champignon) Agaricus hortensis (Agaricus spp.). f213 Rabbit Oryctolagus cuniculus (Oryctolagus spp.). f214 Spinach Spinacia oleracea. f215 Lettuce Lactuca sativa. f216 Cabbage Brassica oleracea var. capitata. f217 Brussels sprouts Brassica oleracea var. gem. f218 Paprika, sweet pepper Capsicum annuum. f219 Fennel seed Foeniculum vulgare. f219 Sage Salvia officinalis. f220 Cinnamon Cinnamomum spp. f221 Coffee Coffea spp. f222 Tea Camellia sinensis. f223 Green olive Olea europaea. f225 Summer squash, pumpkin Cucurbita pepo. f225 Pumpkin Cucurbita maxima. f226 Pumpkin seed Cucurbita pepo. f227 Sugar-beet seed Beta vulgaris. f229 Safflower Seed Carthamus tinctorius. f231 Milk, boiled Bos domesticus (Bos taurus; Bos spp.). f234 Vanilla Vanilla planifolia. f237 Apricot Prunus armeniaca. f241 Gouda cheese NA. f242 Cherry Prunus avium. f244 Cucumber Cucumis sativus. f246 Guar, guar gum Cyamopsis tetragonoloba. f247 Honey NA. f248 Rosemary Rosmarinus officinalis. f254 Plaice Pleuronectes platessa. f255 Plum Prunus domestica ,Prunus americana. f258 Squid Loligo spp. f259 Grape Vitis vinifera (Vitis spp.). f260 Broccoli Brassica oleracea var. italica (Brassica oleracea var. cultivar ). f261 Asparagus Asparagus officinalis. f262 Aubergine, eggplant Solanum melongena. f263 Green pepper Piper nigrum ,Capsicum annuum. f264 Eel Anguilla anguilla. f265 Caraway Carum carvi. f265 Cumin Cuminum cyminum. f266 Mace Myristica fragrans. f267 Cardamon Elettaria cardamomum. f268 Clove Syzygium aromaticum. f269 Basil Ocimum basilicum. f270 Ginger Zingiber officinale. f271 Anise Pimpinella anisum. f272 Tarragon Artemisia dracunculus. f273 Thyme Thymus vulgaris. f274 Marjoram Origanum majorana. f275 Lovage Levisticum officinale. f276 Fennel, fresh Foeniculum vulgare. f277 Dill Anethum graveolens. f278 Bay leaf Laurus nobilis. f279 Chili pepper Capsicum frutescens. f280 Black pepper Piper nigrum. f281 Curry (Santa Maria) NA. f282 Nutmeg Myristica fragrans. f283 Oregano Origanum vulgare. f284 Turkey meat Meleagris gallopavo. f285 Elk/moose meat Alces spp. f286 Mare's milk Equus caballus (Equus spp.). f287 Red kidney bean Phaseolus vulgaris. f288 Blueberry Vaccinium myrtillus (Vaccinium spp.). f289 Date Phoenix dactylifera. f291 Cauliflower Brassica oleracea var. botrytis. f292 Guava Psidium guajava. f293 Papaya Carica papaya. f294 Passion fruit, Maracuja Passiflora edulis (Passiflora spp.). f295 Carambola Averrhoa carambola. f296 Carob Ceratonia siliqua. f297 Gum Arabic Acacia senegal (Acacia spp.). f298 Tragacanth Astragalus spp. f299 Sweet chestnut (chestnut) Castanea sativa. f300 Pinto bean Phaseolus spp. f301 Persimmon (kaki fruit, sharon) Diospyros kaki. f302 Mandarin (tangerine, clementine, satsumas) Citrus reticulata. f305 Fenugreek Trigonella foenum-graecum. f306 Lime Citrus aurantifolia. f307 Hake Merluccius merluccius. f308 Sardine (pilchard) Sardina pilchardus. f310 Blue vetch Lathyrus sativus. f311 Megrim Lepidorhombus whiffiagonis. f315 Green bean Phaseolus vulgaris. f316 Rape seed Brassica napus. f317 Coriander Coriandrum sativum. f318 Jack fruit Artocarpus heterophyllus. f319 Beetroot Beta vulgaris. f320 Crayfish Astacus astacus. f321 Horse meat Equus caballus (Equus spp.). f322 Red currant Ribes sylvestre. f324 Hop (fruit cone) Humulus lupulus. f325 Saffron Colchicum autumnale. f328 Fig Ficus carica. f329 Watermelon Citrullus lanatus. f330 Rose hip Rosa spp. f331 Saffron Crocus sativus. f332 Mint Mentha piperita. f333 Linseed Linum usitatissimum. f336 Jujube Ziziphus jujuba. f336 Wine vinegar Vitis vinifera (Vitis spp.). f337 Sole Solea solea. f337 English sole Parophrys vetulus. f338 Wine, white Vitis vinifera (Vitis spp.). f339 Allspice Pimenta dioica. f339 Wine, red Vitis vinifera (Vitis spp.). f341 Cranberry Vaccinium oxycoccus ,Vaccinium macrocarpon. f342 Olive (black, fresh) Olea europaea. f343 Raspberry Rubus idaeus. f344 Sage Salvia officinalis. f346 Chives Allium schoenoprasum. f347 Quinoa Chenopodium quinoa. f348 Litchi Litchi chinensis. f349 Chum salmon roe Oncorhynchus keta. f358 Artichoke Cynara scolymus. f360 Yogurt NA. f368 Black bass Micropterus dolomieu (Micropterus dolomieui ). f374 Karaya gum Sterculia urens. f375 Horseradish Armoracia rusticana. f377 Maple syrup NA. f379 Okra Abelmoschus esculentus. f382 Beet, sugar Beta vulgaris var. altissima. f401 Loquat Eriobotrya japonica. f402 Fig Ficus carica. f403 Brewer's yeast Saccharomyces cerevisiae. f405 Mint Mentha spp. f406 Arugula Eruca vesicaria. House Dust h1 Greer Labs., Inc NA. h2 Hollister-Stier Labs NA. h6 Japan NA. Venoms & Insects i7 Midge Chironomus yoshimatsui. i8 Moth Bombyx mori ,Heterocera spp. i47 Water flea Daphnia spp. i49 Deer fly Chrysops spp. i51 Black ant Camponotus pennsylvanicus. i54 Flea mix (dog/cat), common flea Ctenocephalides spp. i71 Mosquito Aedes communis ,Aedes spp.and Culex spp. i72 Green nimitti Cladotanytarsus lewisi. i73 Blood worm Chironomus thummi ,Chironomusri parius ,Chironomus spp. i75 European hornet Vespa crabro. i76 Berlin beetle Trogoderma angustum. i77 European paper wasp Polistes dominulus. i78 Fly Musca domestica. i80 Bumblebee Bombus pennsylvanicus. i201 Horse bot fly Gasterophilus intestinalis. i202 Grain weevil Sitophilus granarius. i203 Mediterranean flour moth Ephestia kuehniella (Anagasta kuehniella ). i204 Horse fly Tabanus spp. i205 Bumblebee Bombus terrestris. i208 Api m 1.0101 Apis mellifera. a45 Api m 1 Apis mellifera. i209 Ves v 5.0101 Vespula vulgaris. a670 Ves v 5 Vespula vulgaris. i210 Pol d 5.0101 Polistes dominulus. i211 Ves v 1.0101 Vespula vulgaris. i213 Api m 4 Apis mellifera. i214 Api m 2 Apis mellifera. i215 Api m 3 Apis mellifera. i216 Api m 5 Apis mellifera. i217 Api m 10 Apis mellifera. i220 Bla g 1.0101 Blattella germanica. i221 Bla g 2.0101 Blattella germanica. i222 Bla g 5.0101 Blattella germanica. i223 Bla g 7 Blattella germanica. a46 Api m 2 Apis mellifera. Miscellaneous o1 Cotton, crude fibers Gossypium spp. o3 Cotton (treated) Gossypium spp. o70 Seminal fluid Homo sapiens. o71 Staphylococcus aureus Staphylococcus aureus. o72 Pichia pastoris crude extract customer specific Pichia pastoris. o72 Sperm-sediment Homo sapiens. o73 Pichia pastoris crude extr. vector customer specific Pichia pastoris. o74 Pichia pastoris with vector customer specific Pichia pastoris. o201 Tobacco leaf, tobacco dust Nicotiana tabacum. o202 Artemia salina, fish feed Artemia salina. o203 Tetramin, fish feed NA. o207 Daphnia, fish feed Daphnia spp. o211 Mealworm Tenebrio molitor. o212 Streptavidin Streptomyces avidini. o213 MBP (maltose binding protein) Escherichia coli. o214 CCD; MUXF3 from bromelain Ananas comosus. o72 Enterotoxin A (Sta a SEA) Staphylococcus aureus. o73 Enterotoxin B (Sta a SEB) Staphylococcus aureus. Parasites p1 Ascaris Ascaris suum. p2 Echinococcus Echinococcus granulosus. p3 Schistosoma Schistosoma mansoni. p4 Anisakis (Herring Worm) Anisakis simplex (Anisakis spp.). p5 Toxocara canis Toxocara canis. p10 Ani s 3.0101 Anisakis simplex (Anisakis spp.). p11 Ani s 1 Anisakis simplex (Anisakis spp.). Occupational k4 Threshing dust NA. k5 Flax NA. k7 Hay Dust NA. k8 Hop (hops) Humulus lupulus. k12 Grain mill dust NA. k14 Kapok NA. k20 Sheep's wool (treated) (wool) Ovis aries (Ovis spp.). k21 Sheep's wool (Untreated) Ovis aries (Ovis spp.). k23 Straw Dust NA. k33 Oak NA. k70 Green coffee bean Coffea spp. k71 Castor bean Ricinus communis. k72 Ispaghula Plantago psyllium/Plantago ovata. k73 Silk waste NA. k74 Silk Bombyx mori. k75 Isocyanate TDI (Toluene diisocyanate) NA. k76 Isocyanate MDI (Diphenylmethane diisocyanate) NA. k77 Isocyanate HDI (Hexamethylen diisocyanate) NA. k78 Ethylene oxide NA. k79 Phthalic anhydride NA. k80 Formaldehyde/Formalin NA. k81 Ficus Ficus benjamina (Ficus spp.). k83 Cotton seed Gossypium hirsutum. k84 Sunflower seed Helianthus annuus. k85 Chloramin T NA. k86 Trimellitic anhydride, TMA NA. k87 Asp o 21, alpha-amylase Aspergillus oryzae. k89 Orris root Iris florentina. k99 HSA (Human Serum Albumin) (Hom s HSA) Homo sapiens. k201 Car p 1, Papain Carica papaya. k202 Ana c 2, Bromelain Ananas comosus. k204 Maxatase Bacillus licheniformis. k205 Alcalase Bacillus spp. k206 Savinase, Protease 1 (Bac l Subtilisin) Bacillus spp. k208 Gal d 4, Lysozyme Gallus domesticus (Gallus gallus domesticus; Gallus spp.). k209 Hexahydrophtalic anhydrid NA. k210 Maleic anhydride NA. k211 Methyltetrahydrophtalic anhydrid NA. k212 Abachi wood dust Triplochiton scleroxylon. k213 Pepsin (Sus s Pepsin) Sus scrofa (Sus scrofa domesticus; Sus spp.). k213 TCPA NA. k214 Bougainvillea Bougainvillea spp. k225 Horse radish peroxidase (Arm r HRP) Armoracia rusticana. k226 Ascorbate oxidase (Cuc p ascorbate oxidase) Cucurbita pepo. k301 Flour dust Triticum spp. k501 Savinase customer specific Proprietary knowledge of customer. k502 Lipolase customer specific Proprietary knowledge of customer. k503 Termamyl customer specific Proprietary knowledge of customer. k504 Clazinase customer specific Proprietary knowledge of customer.

Predicate Devices

• Immco Anti-Gliadin Antibody (AGA) IgA ELISA (IgA-AGA) (K964341)
• Immco Anti-Gliadin Antibody (AGA) IgG ELISA (IgG-AGA) (K964344)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM

A. 510(k) Number:
K163177

B. Purpose for Submission:
New device

C. Measurand:
IgA autoantibodies specific for gliadin
IgG autoantibodies specific for gliadin

D. Type of Test:
Immunoassay, semi-quantitative or qualitative

E. Applicant:
IMMCO Diagnostics, Inc.

F. Proprietary and Established Names:
ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA
ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA

G. Regulatory Information:
1. Regulation section:
21 CFR §866.5750, Radioallergosorbent (RAST) immunological test system
2. Classification:
Class II
3. Product code:
MST – Antibodies, Gliadin
4. Panel:
Immunology (82)

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2

H. Intended Use:

1. Intended uses:

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

Enzyme linked immunosorbent assays (ELISA) for the qualitative or semi-quantitative detection of IgA anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease or dermatitis herpetiformis in conjunction with other laboratory and clinical findings.

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

Enzyme linked immunosorbent assays (ELISA) for the qualitative or semi-quantitative detection of IgG anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease or dermatitis herpetiformis in conjunction with other laboratory and clinical findings.

2. Indication(s) for use:

Same as intended use

3. Special conditions for use statement(s):

For prescription use only.

4. Special instrument requirements:

An ELISA microplate reader capable of reading absorbance values at 450 nm. If a dual wavelength microplate reader is available, the reference filter should be set at 450/650 nm. An automatic microplate washer capable of accurately dispensing 200 μL of fluid is also required.

I. Device Description:

Each kit consists of 12 x 8-antigen coated microwell strips, negative control (1x 1.75 mL), positive control (1x 1.75 mL), five assay calibrators (5x 1.75 mL), horseradish peroxidase anti-human IgA or IgG conjugate (1x 15 mL), TMB enzyme substrate (1x 15 mL), stop solution (1x 15 mL), wash buffer (2 vials) and diluent (1x 60 mL). The results are read by a spectrophotometer at 450 nm. Results are expressed in ELISA units per milliliter (EU/mL) and reported as positive or negative.

J. Substantial Equivalence Information:

1. Predicate device names and 510(k) numbers:

Immco Anti-Gliadin Antibody (AGA) IgA ELISA (IgA-AGA), K964341
Immco Anti-Gliadin Antibody (AGA) IgG ELISA (IgG-AGA), K964344

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2. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | New Device ImmuLisa™ Enhanced Gliadin (IgA or IgG) Antibody ELISA | Predicate Immco Anti-Gliadin Antibody (IgA-AGA or IgG-AGA) ELISA |
| Intended Use | Enzyme linked immunosorbent assays (ELISA) for the detection and semi-quantitation of IgA or IgG anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease or dermatitis herpetiformis in conjunction with other laboratory and clinical findings. | Enzyme linked immunosorbent assays (ELISA) for the detection and semi-quantitation of IgA or IgG anti-gliadin antibodies in human serum to aid in the diagnosis of patients with celiac disease and dermatitis herpetiformis. |
| Assay Type | Manual ELISA | Same |
| Solid Phase | Polystyrene microplate wells | Same |
| Capture Antigen | Purified native gliadin antigen | Same |
| Sample Type | Serum | Same |
| Assay Format | Semi-quantitative and qualitative | Same |
| Cutoff | 20 EU/mL | Same |
| Component set | Includes positive control, negative control, calibrators, conjugate, substrate, diluent, wash buffer, stop solution, microplate well. | Same |
| Incubation Times | Positive and negative controls, diluted patient samples: 30 min. Conjugate: 30 min. Enzyme Substrate: 30 min (in the dark). | Same |
| Reaction Temperature | Room temperature | Same |
| Traceability | International Reference Preparation is not available. Results are traceable to in-house standards. | Same |
| Positive Control | Gliadin IgA or Gliadin IgG antibodies | Same |
| Signal | Optical density | Same |
| Instrumentation | Microwell plate reader | Same |
| Storage | 2-8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Device ImmuLisa™ Enhanced Gliadin (IgA or IgG) Antibody ELISA | Predicate Immco Anti-Gliadin Antibody (IgA-AGA or IgG-AGA) ELISA |
| Calibrators | Set of five: Values in EU/mL: 160, 80 40, 20, 1 | Set of four: Varying EU/mL by kit lot |
| Labeled Detection Antibody (Conjugate) | Horseradish peroxidase conjugated to goat anti-human IgA or IgG | Alkaline phosphatase conjugated to goat anti-human IgA or IgG |

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| Enzyme Substrate | Tetramethylbenzidene | p-Nitrophenyl phosphate |
| --- | --- | --- |
| Stop Solution | Sulfuric acid (H2SO4) | Ethylenediaminetetraacetic acid (EDTA) |
| Wash Buffer | Powdered or optional liquid concentrate | Liquid concentrate |
| Linear Range | IgA 7.5 EU/mL – 160 EU/mL
IgG 4.6 EU/mL – 160 EU/mL | Not specified |
| Limit of Detection | IgA 2.6 EU/mL
IgG 2.8 EU/mL | Not specified |
| Sample dilution | 1:101 | 1:51 |
| Reading on spectrophotometer | 450 nm | 405 nm |
| Results Interpretation | Negative: < 20 EU/mL
Indeterminate: 20–25 EU/mL
Positive: > 25 UE/mL | Adults:
Both IgA and IgG
Negative: ≤ 20 EU/mL
Positive: > 20 EU/mL

Children:
IgA
Negative: ≤ 23 EU/mL
Positive: > 23 EU/mL

IgG
Negative: ≤ 28 EU/mL
Positive: > 28 EU/mL |

K. Standard/Guidance Document Referenced:

CLSI guideline EP05-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline-Second Edition"

CLSI guideline EP06-A, "Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline"

CLSI guideline EP07-A2, "Interference Testing in Clinical Chemistry; Approved Guideline–Second Edition"

CLSI guideline EP09-A2, "Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline."

CLSI guideline EP12-A2, "User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline–Second Edition"

CLSI guideline EP17-A2, "Evaluation of Detection Capability for Clinical. Laboratory Measurement Procedures; Approved Guideline–Second Edition"

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L. Test Principle:

The test is performed as a solid phase immunoassay. Gliadin antigen is bound to the wells of a polystyrene microwell plate followed by blocking the unreacted sites to reduce non-specific binding. Controls, calibrators and patient sera are added to separate wells, allowing any gliadin antibodies present to bind to the immobilized antigen. Unbound antibodies and other serum proteins are removed by washing the microplate wells. Bound antibodies are detected by adding an enzyme labeled anti-human IgA or IgG conjugate to the microplate wells. These enzyme conjugated antibodies bind specifically to the human immunoglobulin of the appropriate class. After washing away any unbound conjugate, specific enzyme substrate (TMB) is then added to the wells and the presence of antibodies is detected by a color change produced by the conversion of TMB substrate to a colored reaction product. After stopping the enzymatic reaction, the intensity of color change, which is proportional to the concentration of antibody, is read by a spectrophotometer at 450 nm. Results are expressed in ELISA units per milliliter (EU/ml) and reported as positive or negative.

Semi-quantitative results are determined from a series of five calibrators (160 EU/mL, 80 EU/mL, 40 EU/mL, 20 EU/mL, and 1 EU/mL). Values less than 20 EU/mL are considered negative results while values greater than 25 EU/mL are considered positive; results between 20 EU/mL and 25 EU/mL are considered 'indeterminate/borderline'. The sponsor states the following recommendations in the Package Insert: "Indeterminate/borderline results should be retested and evaluated along with other laboratory methods, such as assays for detection of EMA and/or tTG antibodies".

Qualitative results are determined using a ratio of the absorbance of the sample to the absorbance of the cut-off calibrator (20 EU/mL). The ratio is multiplied by the concentration of the cut-off calibrator to give a numerical value. Values greater than or equal to 20 EU/mL are considered positive and &lt;20 EU/mL values are considered negative.

M. Performance Characteristics:

1. Analytical performance:

a. Precision/Reproducibility:

Semi-Quantitative Precision:

Precision performance was evaluated in accordance with CLSI guideline EP05-A2 "Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline". A panel consisting of seven patient sera with levels of gliadin antibodies that cover the analytical measuring range was assayed in duplicate, twice a day, for 20 days with one reagent lot (a total of 80 replicates per sample). Testing was performed by two operators using different equipment sets. The first operator performed the assay with a multi-channel pipettor (BioHit), microplate washer (ELx405) and microplate reader (ELx808). The second operator used different instruments from the same type of equipment. All %CV values were within the manufacturer's pre-determined acceptance limit. The results are summarized in the table below for each assay:

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ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| Specimen | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Runs | | Between-Days | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 3.6 | 0.4 | 9.9 | 0.1 | 3.1 | 0.2 | 6.6 | 0.4 | 10.3 |
| 2 | 15.3 | 0.8 | 5.3 | 0.3 | 7.1 | 0.6 | 3.8 | 0.9 | 5.6 |
| 3 | 20.2 | 0.8 | 4.0 | 0.4 | 2.1 | 0.8 | 3.9 | 0.9 | 4.5 |
| 4 | 23.7 | 1.0 | 4.1 | 0.9 | 3.9 | 0.8 | 3.3 | 1.4 | 5.7 |
| 5 | 67.9 | 1.8 | 2.6 | 1.3 | 1.9 | 1.3 | 1.9 | 2.2 | 3.2 |
| 6 | 93.2 | 4.9 | 5.2 | 2.1 | 2.2 | 4.8 | 5.1 | 5.6 | 5.7 |
| 7 | 143.5 | 9.3 | 6.4 | 4.4 | 3.1 | 10.3 | 7.2 | 11.0 | 7.1 |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| Specimen | Mean (EU/mL) | Within-Run (Repeatability) | | Between-Runs | | Between-Days | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 1 | 13.7 | 0.6 | 4.1 | 1.1 | 8.0 | 0.6 | 4.2 | 1.4 | 9.9 |
| 2 | 17.4 | 0.6 | 3.3 | 0.9 | 5.4 | 0.3 | 1.8 | 1.1 | 6.5 |
| 3 | 19.9 | 0.7 | 3.3 | 0.7 | 3.6 | 0.7 | 3.2 | 1.2 | 5.9 |
| 4 | 23.4 | 0.7 | 2.8 | 1.0 | 4.4 | 0.5 | 2.2 | 1.2 | 5.3 |
| 5 | 44.8 | 1.8 | 4.0 | 1.6 | 3.5 | 0.7 | 1.6 | 2.5 | 5.5 |
| 6 | 96.8 | 3.5 | 3.6 | 7.6 | 7.8 | 1.7 | 1.7 | 8.5 | 8.8 |
| 7 | 153.4 | 6.6 | 4.3 | 11.6 | 7.6 | 5.1 | 3.3 | 13.4 | 8.7 |

## Qualitative Reproducibility:

Studies were performed under the guidance of CLSI EP12-A2 'User Protocol for Evaluation of Qualitative Test Performance.' Eighty replicates of each patient serum in the negative range, ~20% below cut-off, at ~cut-off, ~20% above cut-off and in the moderate positive range of the assays were performed to determine intra-assay qualitative reproducibility. Results were calculated using single-point (qualitative) analysis as indicated in the product insert. All values for qualitative agreement were within the manufacturer's pre-determined acceptance limit. Results are summarized below.

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| Specimen | Mean (EU/mL) | Total replicates | Qualitative Agreement | |
| --- | --- | --- | --- | --- |
| | | | Positive/negative | % |
| Negative | 2.6 | 80 | 0/80 | 100 |
| Cut-off -20% | 15 | 80 | 0/80 | 100 |
| Cut-off | 20.2 | 80 | 41/39 | 51.3 |
| Cut-off +20% | 24.3 | 80 | 80/0 | 100 |
| Moderate Positive | 67.5 | 80 | 80/0 | 100 |
| Moderate to High Positive | 83.1 | 80 | 80/0 | 100 |
| High Positive | 102.8 | 80 | 80/0 | 100 |

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ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| Specimen | Mean (EU/mL) | Total replicates | Qualitative Agreement | |
| --- | --- | --- | --- | --- |
| | | | Positive/negative | % |
| Negative | 13.3 | 80 | 0/80 | 100 |
| Cut-off –20% | 17.1 | 80 | 0/80 | 100 |
| Cut-off | 19.7 | 80 | 46/34 | 57.5 |
| Cut-off +20% | 23.4 | 80 | 80/0 | 100 |
| Moderate Positive | 51.7 | 80 | 80/0 | 100 |
| Moderate to High Positive | 64.7 | 80 | 80/0 | 100 |
| High Positive | 82.9 | 80 | 80/0 | 100 |

Lot-to-lot Reproducibility:

To evaluate lot-to-lot reproducibility, a panel of five samples with levels of gliadin antibodies spanning the assay range was tested in replicates of five on three different lots over the course of five days. All %CV values were within the manufacturer's pre-determined acceptance limit. The results are summarized in the table below for each assay:

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| Specimen | Mean (EU/mL) | Within Run | | Between Lots | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 9.9 | 0.4 | 3.7 | 0.7 | 7.5 | 0.7 | 7.5 |
| 2 | 22.6 | 1.4 | 6.0 | 1.7 | 7.5 | 1.8 | 7.9 |
| 3 | 35.2 | 0.8 | 2.3 | 2.4 | 6.7 | 2.4 | 6.7 |
| 4 | 77.9 | 1.6 | 2.1 | 5.7 | 7.3 | 5.9 | 7.6 |
| 5 | 149.7 | 6.6 | 4.4 | 10.7 | 7.2 | 11.7 | 7.8 |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| Specimen | Mean (EU/mL) | Within Run | | Between Lots | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 16.13 | 0.6 | 3.7 | 1.5 | 9.2 | 1.5 | 9.2 |
| 2 | 22.59 | 1.0 | 4.5 | 2.1 | 9.1 | 2.1 | 9.1 |
| 3 | 35.48 | 1.2 | 3.3 | 3.1 | 8.7 | 3.1 | 8.7 |
| 4 | 87.31 | 2.7 | 3.1 | 5.4 | 6.2 | 5.7 | 6.5 |
| 5 | 122.87 | 5.0 | 4.0 | 10 | 8.1 | 11.3 | 9.2 |

Site-to-site Reproducibility:

To evaluate site-to-site reproducibility, a panel of five samples with levels of gliadin antibodies spanning the assay range was tested in replicates of five at three different sites over the course of five days using one lot of reagent. All %CV values were within the manufacturer's pre-determined acceptance limit. The results are summarized in the table below for each assay:

{7}

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| Specimen | Mean (EU/mL) | Within Run | | Between Sites | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 10.0 | 0.4 | 4.4 | 0.8 | 7.6 | 0.8 | 8.3 |
| 2 | 22.7 | 0.6 | 2.7 | 1.4 | 6.2 | 2.1 | 9.4 |
| 3 | 47.7 | 1.4 | 2.9 | 2.9 | 6.0 | 4.1 | 8.6 |
| 4 | 88.4 | 4.7 | 5.3 | 7.8 | 8.8 | 8.5 | 9.6 |
| 5 | 146.2 | 5.1 | 3.5 | 12.0 | 8.2 | 12.8 | 8.8 |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| Specimen | Mean (EU/mL) | Within Run | | Between Sites | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| 1 | 14.0 | 0.7 | 5.1 | 0.9 | 6.4 | 1.3 | 9.1 |
| 2 | 24.1 | 1.0 | 4.0 | 1.9 | 7.9 | 2.1 | 8.5 |
| 3 | 33.9 | 1.4 | 4.0 | 2.4 | 7.0 | 2.4 | 7.0 |
| 4 | 70.0 | 2.9 | 4.1 | 5.1 | 7.3 | 5.8 | 8.2 |
| 5 | 151.1 | 5.3 | 3.5 | 10.8 | 7.2 | 14.9 | 9.2 |

b. Linearity/assay reportable range:

Linearity and recovery were tested by diluting positive specimens across the measuring range in equidistant dilutions with negative patient sera. The observed values were graphed against the calculated values and a linear regression was performed. Results summarized in the table below were within the manufacturer's pre-determined acceptance criteria of recovery.

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| Specimen | Dilution Range (EU/mL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery |
| --- | --- | --- | --- | --- | --- |
| B2GPI IgG Qualitative Precision | 1.01 (0.92 to 1.10) | 1.13 (-1.14 to 3.39) | 0.99 | 92% | 94% |
| 2 | 2.3 to 97.2 | 1.04 (0.95 to 1.12) | 1.45 (-3.18 to 6.08) | 0.99 | 90% to 96% |
| 3 | 4.7 to 127.4 | 0.85 (0.79 to 0.91) | 2.06 (-3.39 to 7.41) | 1.0 | 102% to 117% |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| Specimen | Dilution Range (EU/mL) | Slope (95% CI) | Y-Intercept (95% CI) | R² | % Recovery |
| --- | --- | --- | --- | --- | --- |
| 1 | 9.0 to 47.0 | 0.98 (0.88 to 1.08) | 0.87 (-2.16 to 3.90) | 0.99 | 93% to 106% |
| 2 | 12.7 to 116.8 | 0.98 (0.91 to 1.05) | -1.47 (-6.66 to 3.73) | 1.0 | 102% to 110% |
| 3 | 58.1 to 147.4 | 0.98 (0.87 to 1.08) | 3.67 (-7.70 to 15.04) | 0.99 | 98% to 106% |

{8}

The linear range was determined to be 7.5–160 EU/mL for the gliadin IgA assay and 4.6–160 EU/mL for the gliadin IgG assay. For test results above 160 EU/mL, the package insert recommends dilution and retesting of the samples.

High dose hook effect: Four high concentrations specimens were serially diluted 1:100 to assess the presence of any artefactual decrease in assay signal associated with gliadin antibody excess (hook effect). No hook effect was demonstrated in dilution sample levels up to 5515.8 EU/mL of gliadin IgA and 1686.3 EU/mL of gliadin IgG.

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

i. Traceability:

There are currently no recognized international standards for the measurement of gliadin antibodies. Calibrator and Control values are directly traceable to in-house standards.

ii. Value Assignment:

Calibrators and positive controls are dilutions of pooled gliadin antibody positive sera obtained from various commercial plasma centers. The calibrators and controls are taken from different pooled sera. In order to have traceability with newly made calibrators, each new lot of calibrators is assayed on an already cleared kit and compared to the existing calibrators as reference. Each lot of calibrator is also tested in comparison with normal human sera, clinical samples and internal standards. Five calibrator levels (Cal A-E) with assigned values from 0–160 EU/mL are included to provide semi-quantitation and must be used with each run. The cut-off calibrator for the qualitative analysis is derived from Cal D. Test results greater than or equal to Cal D are considered positive.

For semi-quantitative determinations the positive controls must give values in the range stated on the vial. The negative control contains negative human serum in buffer. The negative control must be &lt;10 EU/mL.

iii. Stability:

Shelf-life Stability:

Accelerated and real-time stability studies for each ImmuLisa™ Enhanced Gliadin Antibody ELISA were conducted on three lots of components/reagents. The accelerated stability study was conducted with materials incubated at 37°C. Data from the accelerated stability study set the shelf-life stability at 18 months. A real-time stability study is on-going and currently supports an 18-month shelf-life stability claim.

Open Kit Stability:

For the open kit stability study, materials in each ImmuLisa™ Enhanced Gliadin Antibody ELISA are opened and stored in a dark environment as required for bench-top usage, then assayed at 15, 45 and 90 day intervals. Data from the open kit stability study demonstrate that

9

{9}

opened reagents are stable at 45 days, but the sponsor chose a more restrictive one month open kit stability claim.

Sample Stability and Storage:

The package insert recommends that sera be assayed soon after separation or stored in aliquots at $2 - 8^{\circ}\mathrm{C}$ for no longer than one week. For longer storage, serum specimens should be frozen. Avoid repeated freezing and thawing of samples. It is recommended that frozen specimens be tested within one year.

# d. Detection limit:

The analytical sensitivity was determined in accordance with CLSI EP17-A2. The Limit of Blank (LoB) was determined by assaying a blank sample in 60 replicates. The limit of detection (LoD) was determined by assaying six low negative sera in ten replicates over three days using two reagent lots of each ImmuLisa™ Enhanced Gliadin Antibody ELISA. The Limit of Quantitation was determined by testing four low level samples in replicates of four over three days using two reagent lots. The claimed LoB, LoD and LoQ are summarized in the table below:

| ImmuLisa™ Gliadin Antibody ELISAs | LoB (EU/mL) | LoD (EU/mL) | LoQ (EU/mL) |
| --- | --- | --- | --- |
| Gliadin IgA | 2.2 | 3.6 | 7.5 |
| Gliadin IgG | 1.6 | 2.8 | 4.6 |

# e. Analytical specificity:

# i. Endogenous Interference:

Interference studies were performed according to CLSI EP07-A2 by testing five serum samples with gliadin antibody levels corresponding to the negative range, near the assay cutoff and in the low, moderate and high positive range. Each sample was mixed with known quantities of potentially interfering substances and analyzed in one assay run with two replicates on one kit lot of each assay. The recovery was calculated by comparing to control samples spiked with the same volume of diluents. No significant interference was demonstrated in the ImmuLisa™ Enhanced Gliadin Antibody ELISAs with the following substances up to the concentrations listed in the table below:

| Potential Interfering Compound | Test Concentration |
| --- | --- |
| Hemoglobin | 2 g/L |
| Bilirubin | 342 μmol/L |
| Triglycerides | 37 mmol/L |
| Rheumatoid Factor | 100 EU/mL |
| Total cholesterol | 13 mmol/L |

{10}

The 'Limitations of Procedure' section of the Package Insert states "This test should not be performed on grossly hemolyzed, microbially contaminated or lipemic samples".

ii. Cross-reactivity:

Numerous potentially cross-reactive autoimmune and infectious disease sera were tested for gliadin antibody levels. Refer to test results for 456 serum samples from patients in the Non-Target Disease Group in the table presented in the section below on Clinical studies. The number of serum samples tested positive is 12 (2.4%) for gliadin IgA and 21 (4.6%) for gliadin IgG. The 'Limitations of Procedure' section of the Package Insert states: "The results obtained serve only as an aid in the diagnosis and should not be interpreted as diagnostic in themselves."

f. Assay cut-off:

The assay cut-offs for the ImmuLisa™ Enhanced Gliadin Antibody ELISAs were determined by testing specimens from 126 healthy blood donors. Using the 97.5th percentile value of the results obtained, an arbitrary value of 20 EU/mL was assigned for the cut-off.

| Gliadin antibody value | Result interpretation |
| --- | --- |
| <20 EU/mL | Negative |
| 20–25 EU/mL | Indeterminate (Borderline) |
| >25 EU/mL | Positive |

Utilizing the 20 EU/mL cut-off value, 4.2% (5/120) and 1.7% (2/120) of the samples from healthy blood donors and patients in the Non-Target Disease Group were positive for gliadin IgA and gliadin IgG, respectively

2. Comparison studies:

a. Method comparison with predicate devices:

For analysis of agreement with a predicate device, clinically defined serum samples from the target disease group and non-target disease group were assayed for each gliadin antibody on both ImmuLisa™ Enhanced Gliadin Antibody ELISA and Anti-Gliadin (AGA) ELISA. The diagnosed conditions in each group and the numbers of specimens tested for each gliadin antibody are presented in the table below:

| | Gliadin IgA | Gliadin IgG |
| --- | --- | --- |
| Target Disease Group | | |
| Celiac Disease (CD) | 135 | 136 |
| IgA Deficient CD (Adult) | 23 | 24 |
| IgA Deficient CD (Pedriatic) | 11 | 22 |
| Pediatric Celiac Disease | 35 | 37 |
| Dermatitis Herpetiformis | 26 | 27 |
| Total | 230 | 246 |

{11}

| Non-Target Disease Group | | |
| --- | --- | --- |
| Systemic Lupus Erythematosus | 21 | 23 |
| Rheumatoid Arthritis | 30 | 30 |
| Systemic Sclerosis | 12 | 15 |
| Hashimoto's Thyroiditis | 4 | 7 |
| Graves's Disease | 6 | 5 |
| Crohn's Disease | 14 | 13 |
| Ulcerative Colitis | 8 | 15 |
| Toxoplasmosis | 7 | 13 |
| Cytomegalovirus | 11 | 11 |
| Herpes Simplex Virus 1 | 8 | 12 |
| Herpes Simplex Virus 2 | 8 | 13 |
| Rubella | 8 | 15 |
| Lyme Disease | 9 | 13 |
| Helicobacter pylori | 24 | 28 |
| Total | 170 | 213 |

Only specimens in the linear range of the assays were included in the method comparison. Greater than $15\%$ of the samples fell near the cutoff of both assays. The results for each assay are summarized below:

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| | AGA IgA ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA (EU/mL) | Positive: >25 | 96 | 17 | 113 |
| | Equivocal: 20–25 | 10 | 12 | 22 |
| | Negative: <20 | 3 | 262 | 265 |
| Total | | 109 | 291 | 400* |
| * target disease group (n=230) and non-target disease group (n=170) | | | | |

Agreements were calculated by grouping ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA indeterminate results (20-25 EU/mL) with its test negative results, and then agreements were calculated again by grouping Indeterminate results with the test positive results:

Indeterminate gliadin IgA results considered as negative:

| | AGA IgA ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA (EU/mL) | Positive: >25 | 96 | 17 | 113 |
| | Negative: ≤25 | 13 | 274 | 287 |
| Total | | 109 | 291 | 400 |
| Positive percent agreement: | | 88.1% (96/109) | 95% CI: 80.1%-93.2% | |
| Negative percent agreement: | | 94.2% (274/291) | 95% CI: 90.6%-96.5% | |
| Total percent agreement: | | 92.5% (370/400) | 95% CI: 89.5%-94.7% | |

{12}

Indeterminate gliadin IgA results considered as positive:

| | AGA IgA ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA (EU/mL) | Positive: ≥20 | 106 | 29 | 135 |
| | Negative: <20 | 3 | 262 | 265 |
| Total | | 109 | 291 | 400 |
| Positive percent agreement: 97.2% (106/109) 95% CI: 91.6%-99.3% | | | | |
| Negative percent agreement: 90.0% (262/291) 95% CI: 85.9%-93.1% | | | | |
| Total percent agreement: 92.0% (368/400) 95% CI: 88.9%-94.3% | | | | |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| | AGA IgG ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA (EU/mL) | Positive: >25 | 190 | 10 | 200 |
| | Equivocal: 20–25 | 9 | 4 | 13 |
| | Negative: <20 | 6 | 240 | 246 |
| Total | | 205 | 254 | 459* |
| * target disease group (n=246) and non-target disease group (n=213) | | | | |

Agreements were calculated by grouping ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA indeterminate results (20-25 EU/mL) with its test negative results, and then agreements were calculated again by grouping indeterminate results with the test positive results:

Indeterminate gliadin IgG results considered as negative:

| | AGA IgG ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA (EU/mL) | Positive: >25 | 190 | 10 | 200 |
| | Negative: ≤25 | 15 | 244 | 259 |
| Total | | 205 | 254 | 459 |
| Positive percent agreement: 92.7% (190/205) 95% CI: 88.0%-95.7% | | | | |
| Negative percent agreement: 96.1% (244/254) 95% CI: 92.7%-98.0% | | | | |
| Total percent agreement: 94.6% (444/459) 95% CI: 92.1%-96.3% | | | | |

Indeterminate gliadin IgG results considered as positive:

| | AGA IgG ELISA (EU/mL) | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive: >20 | | Negative: ≤20 |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA (EU/mL) | Positive: >20 | 199 | 14 | 203 |
| | Negative: ≤20 | 6 | 240 | 246 |
| Total | | 205 | 254 | 459 |
| Positive percent agreement: 97.1% (199/205) 95% CI: 93.4%-98.8% | | | | |
| Negative percent agreement: 94.5% (240/254) 95% CI: 90.7%-96.8% | | | | |
| Total percent agreement: 95.6% (439/459) 95% CI: 93.4%-97.2% | | | | |

{13}

b. Matrix comparison:

Not applicable since human serum is the only claimed specimen matrix. The 'Specimen Collection and Handling' section in the Package Insert states "Only serum specimens should be used in this procedure".

3. Clinical studies:

a. Clinical sensitivity and specificity:

A total of 751 serum samples were included in the clinical validation for the ImmuLisa™ Enhanced Gliadin Antibody ELISAs. The validation set of samples includes a target disease group (250 celiac disease and 45 dermatitis herpetiformis) and a non-target disease group (456 samples from patients with autoimmune and infectious conditions expected to be found in the intended use population). Test results for each disease group are shown below:

| | Gliadin IgA | | Gliadin IgG | | |
| --- | --- | --- | --- | --- | --- |
| | | # Pos* | % Pos | # Pos* | % Pos |
| Target Disease Group | | | | | |
| Celiac Disease | 200 | 130 | 65 | 154 | 77 |
| Pediatric Celiac Disease | 50 | 34 | 68 | 37 | 74 |
| Dermatitis Herpetiformis | 45 | 16 | 35.6 | 26 | 57.8 |
| Total | 295 | 180 | 50.1 | 217 | 72.5 |
| Non-Target Disease Group | | | | | |
| Systemic Lupus Erythematosus | 30 | 2 | 6.7 | 3 | 10 |
| Granulomatosis with Polyangiitis | 30 | 0 | 0 | 1 | 0 |
| Rheumatoid Arthritis | 30 | 1 | 3.3 | 0 | 0 |
| Systemic Sclerosis | 30 | 0 | 0 | 0 | 0 |
| Hashimoto's Thyroiditis | 15 | 0 | 0 | 1 | 6.7 |
| Graves's Disease | 15 | 1 | 6.7 | 0 | 0 |
| Crohn's Disease | 25 | 1 | 4 | 2 | 8 |
| Ulcerative Colitis | 25 | 0 | 0 | 1 | 4 |
| Autoimmune Hepatitis | 30 | 0 | 0 | 1 | 3.3 |
| Primary Biliary Liver Cirrhosis | 50 | 4 | 8 | 3 | 6 |
| Toxoplasmosis | 20 | 0 | 0 | 0 | 10 |
| Cytomegalovirus | 20 | 0 | 0 | 0 | 0 |
| Herpes Simplex Virus 1 | 20 | 0 | 0 | 0 | 0 |
| Herpes Simplex Virus 2 | 20 | 0 | 0 | 3 | 15 |
| Lyme's Disease | 36 | 0 | 0 | 3 | 8.3 |
| Rubella | 20 | 0 | 0 | 0 | 0 |
| Helicobacter pylori | 40 | 3 | 7.5 | 3 | 7.5 |
| Total | 456 | 12 | 2.4 | 21 | 4.6 |
| Pos: positive
* Indeterminate samples were considered positive | | | | | |

{14}

The performance of each ImmuLisa™ Enhanced Gliadin Antibody ELISA was evaluated against the clinical diagnosis of celiac disease and dermatitis herpetiformis separately. In both evaluations, the clinical sensitivity and specificity were calculated by grouping the assay's indeterminate results with its test negative results, and then sensitivity and specificity were calculated again by grouping the assay's equivocal results with its test positive results.

The clinical sensitivity and specificity of the ImmuLisa™ Enhanced Gliadin Antibody ELISAs in this sample cohort are summarized in the following tables for celiac disease:

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 139 | 7 | 146 |
| | Equivocal | 25 | 5 | 30 |
| | Negative | 86 | 444 | 530 |
| Total | | 250 | 456 | 706* |
| * celiac disease (n=200), pediatric celiac disease (n=50) and non-target disease group (n=456) | | | | |

Indeterminate gliadin IgA results considered as negative:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 139 | 7 | 146 |
| | Negative | 111 | 449 | 560 |
| Total | | 250 | 456 | 706 |
| Sensitivity: 55.6% (139/250) 95% CI: 49.2%-61.8%
Specificity: 98.5% (449/456) 95% CI: 96.7%-99.3% | | | | |

Indeterminate gliadin IgA results considered as positive:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 164 | 12 | 176 |
| | Negative | 86 | 444 | 530 |
| Total | | 250 | 456 | 706 |
| Sensitivity: 65.6% (164/250) 95% CI: 59.3%-71.4%
Specificity: 97.4% (444/456) 95% CI: 95.3%-98.6% | | | | |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 171 | 15 | 186 |
| | Equivocal | 20 | 6 | 26 |
| | Negative | 59 | 435 | 494 |
| Total | | 250 | 456 | 706 |

{15}

Indeterminate gliadin IgG results considered as negative:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 171 | 15 | 186 |
| | Negative | 79 | 441 | 520 |
| Total | | 250 | 456 | 706 |
| Sensitivity: 68.4% (171/250) | | 95% CI: 62.2%-74.0% | | |
| Specificity: 96.7% (441/456) | | 95% CI: 94.5%-98.1% | | |

Indeterminate gliadin IgG results considered as positive:

| | Celiac Disease Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 191 | 21 | 176 |
| | Negative | 59 | 435 | 494 |
| Total | | 250 | 456 | 706 |
| Sensitivity: 76.4% (191/250) | | 95% CI: 70.5%-81.4% | | |
| Specificity: 95.4% (435/456) | | 95% CI: 92.9%-97.1% | | |

The clinical sensitivity and specificity of the ImmuLisa™ Enhanced Gliadin Antibody ELISAs in this sample cohort are summarized in the following tables for dermatitis herpetiformis:

ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 15 | 7 | 22 |
| | Equivocal | 1 | 5 | 6 |
| | Negative | 29 | 444 | 473 |
| Total | | 45 | 456 | 501 |
| * dermatitis herpetiformis (n=45) and non-target disease group (n=456) | | | | |

Indeterminate gliadin IgA results considered as negative:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 15 | 7 | 22 |
| | Negative | 30 | 449 | 479 |
| Total | | 45 | 456 | 501 |
| Sensitivity: 33.3% (15/45) 95% CI: 20.4%-49.1%
Specificity: 98.5% (449/456) 95% CI: 96.7%-99.3% | | | | |

{16}

Indeterminate gliadin IgA results considered as positive:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgA Antibody ELISA | Positive | 16 | 12 | 28 |
| | Negative | 29 | 444 | 473 |
| Total | | 45 | 456 | 501 |
| Sensitivity: 35.6% (16/45) | | 95% CI: 22.3%-51.3% | | |
| Specificity: 97.4% (444/456) | | 95% CI: 95.3%-98.6% | | |

ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 25 | 15 | 40 |
| | Equivocal | 1 | 6 | 7 |
| | Negative | 19 | 435 | 454 |
| Total | | 45 | 456 | 501 |

Indeterminate gliadin IgG results considered as negative:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 25 | 15 | 40 |
| | Negative | 20 | 441 | 461 |
| Total | | 45 | 456 | 501 |
| Sensitivity: 55.6% (25/45) | | 95% CI: 40.1%-70.0% | | |
| Specificity: 96.7% (441/456) | | 95% CI: 94.5%-98.1% | | |

Indeterminate gliadin IgG results considered as positive:

| | Dermatitis Herpetiformis Diagnosis | | Total | |
| --- | --- | --- | --- | --- |
| | | Positive | | Negative |
| ImmuLisa™ Enhanced Gliadin IgG Antibody ELISA | Positive | 26 | 21 | 47 |
| | Negative | 19 | 435 | 454 |
| Total | | 45 | 456 | 501 |
| Sensitivity: 57.8% (26/45) | | 95% CI: 42.2%-72.0% | | |
| Specificity: 95.4% (435/456) | | 95% CI: 92.9%-97.1% | | |

4. Clinical cut-off:
Refer to assay cut-off.

{17}

5. Expected values/Reference range:

Test results in a normal population are expected to be negative. However, it has been determined that some apparently healthy, asymptomatic individuals may test positive for gliadin IgA or gliadin IgG (Rashtak S. et al. Clin Gastroenterol Hepatol. 2008; 6:426-432).

A total of 269 normal human sera (130 pediatrics and 139 adults) tested with the ImmuLisa™ Enhanced Gliadin Antibody ELISAs yielded 8 (3%) positive results for gliadin IgA (all adults) and 10 (3.7%) positive for gliadin IgG (4 pediatrics and 6 adults).

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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